Insecticidal protein discovery platform and insecticidal proteins discovered therefrom

ABSTRACT

The disclosure presents a platform for discovering novel insecticidal proteins from highly heterogeneous environmental sources. The methodology utilizes metagenomic enrichment procedures and unique genetic amplification techniques, which enables access to a broad class of unknown microbial diversity and their resultant proteome. The disclosed insecticidal protein discovery platform (IPDP) can be computationally driven and is able to integrate molecular biology, automation, and advanced machine learning protocols. The platform will enable researchers to rapidly and accurately access the vast repertoire of untapped insecticidal proteins produced by uncharacterized and complex microbial environmental samples. Also presented herein are a group of newly discovered pore-forming toxins (PFT) from a rare class of insecticidal proteins, which were discovered utilizing the insecticidal protein discovery platform.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.17/009,438, filed Sep. 1, 2020, which is a Continuation of InternationalPCT Application No. PCT/US2019/020218, filed Mar. 1, 2019, which claimsthe benefit of priority to U.S. Provisional Application No. 62/637,515filed on Mar. 2, 2018, each of which is hereby incorporated by referencein its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is ZYMR_022_01WO_SeqList_ST25.txt. The text file is167 KB, was created on Feb. 27, 2019, and is being submittedelectronically via EFS-Web.

FIELD

The present disclosure is directed to an approach for discovering novelinsecticidal proteins from highly heterogeneous environmental sources.The methodology utilizes metagenomic enrichment procedures and uniquegenetic amplification techniques, which enables access to a broad classof unknown microbial diversity and their resultant proteome.

The disclosed insecticidal protein discovery platform (IPDP) can becomputationally driven and is able to integrate molecular biology,automation, and advanced machine learning protocols. The platform willenable researchers to rapidly and accurately access the vast repertoireof untapped insecticidal proteins produced by uncharacterized andcomplex microbial environmental samples.

Also presented herein are a group of newly discovered pore-formingtoxins (PFT) from a rare class of insecticidal proteins, which werediscovered utilizing the insecticidal protein discovery platform.

BACKGROUND

It is estimated that by the year 2050 the world's population will havereached over 9 billion people. Estimates by agricultural experts at theUnited Nations project that in order to feed such a large globalpopulation, then total food production must increase by 70% to meetfuture demands. This challenge is exacerbated by numerous factors,including: diminishing freshwater resources, limited supplies of arableland, rising energy prices, increasing input costs, and environmentconcerns attached to modern row crop agriculture.

An age old problem, which will continue to be one of the most pressingconcerns facing our global agricultural industry, is pesticidal pressureand the associated reduction in yields and reduced productivity stemmingtherefrom. Traditional synthetic chemicals have been successful inhelping farmers battle problematic insects, but these chemicals faceincreasing scrutiny over concerns about their impact on human health andpotential detrimental environmental effects. Consequently, in order tomeet the needs of a growing global population, there will be anincreased demand for biotechnological solutions to combat agriculturalpests.

One leading biotechnological pesticide solution comes from Bacillusthuringiensis (Bt), a gram-positive, spore forming bacterium. Btbacteria were identified as insect pathogens and their insecticidalactivity was attributed to the parasporal crystals encoded by the Crygenes, of which there are over 100 known isoforms. This observation ledto the development of bioinsecticides based on Bt bacteria for thecontrol of certain insect species. Plants have now been geneticallyengineered to express the Bt insecticidal proteins, which alleviates theneed for external application to the plants. However, similar to thesituation where insect resistance develops due to continuous use ofchemical insecticides, the continuous expression of these insecticidalBt proteins in plants also imposes strong selection for resistance intarget pest populations. Consequently, the industry has seen an alarmingrate of insect populations becoming resistant to Bt crops. Furthermore,Bt proteins have a limited range of activity and are not effectiveagainst some of the currently problematic insect species.

Thus, in view of an expanding global population, environmental concernsassociated with traditional chemical insecticides, and growing insectresistance to Bt traits, there is a great need in the art for theidentification of novel insecticidal proteins, which can be incorporatedinto biotechnological products useful for modern agriculture.

SUMMARY OF THE DISCLOSURE

The present disclosure provides novel insecticidal proteins, which canbe utilized in modern row crop agriculture. These insecticidal proteinscan be developed into standalone products for application directly to aplant species, or can be incorporated into the genome of a host plantfor expression.

Unlike traditional synthetic chemical insecticides, the taughtinsecticidal proteins do not pose environmental concerns. Further, theinsecticidal proteins belong to a newly discovered class, which haveseveral advantages over the current industry standard Cry proteinproducts derived from Bacillus thuringiensis (Bt) encoded sequences.

Besides the novel insecticidal proteins themselves, the disclosureprovides a platform for discovering additional insecticidal proteins, byaccessing the vast repertoire of untapped insecticidal proteins producedby uncharacterized and complex microbial environmental samples.

The insecticidal protein discovery platform (IPDP) utilizes metagenomicenrichment procedures and unique genetic amplification techniques,enabling access to a broad class of unknown microbial diversity andtheir resultant proteome. Because the platform can be computationallydriven and is able to integrate molecular biology, automation, andadvanced machine learning protocols, researchers will now be able torapidly and systematically develop models and search queries, toidentify additional novel insecticidal proteins.

In certain embodiments, the disclosure provide a method for constructinga genomic library, enriched for DNA from Pseudomonas encodinginsecticidal proteins, comprising: a) providing an initial samplecomprising one or more microorganisms; b) exposing the initial sample toa solid nutrient limiting media that enriches for growth of species fromthe genus Pseudomonas, which results in a subsequent sample enriched forPseudomonas sp.; c) isolating DNA from the subsequent enriched sample;d) extracting DNA from the isolated DNA and performing degenerate PCRwith primers selected to amplify target insecticidal protein genes; e)cloning the PCR-amplified DNA into a plasmid; and f) sequencing thecloned DNA from said plasmid. In certain embodiments, the methodcomprises assembling the sequenced DNA into a genomic library. Incertain embodiments, the method comprises identifying insecticidalprotein genes within the sequenced DNA. In some embodiments, theidentified insecticidal proteins are unknown. In some embodiments, aHidden Markov model is used to identify insecticidal protein genes. Insome embodiments, any gene (i.e. a nucleotide sequence) in Table 3 (e.g.SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67,69, and 71) can be found. In some embodiments, any gene encoding aprotein found in Table 3 (e.g. SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, 66, 68, 70, and 72) can be found. In someembodiments, the primers are selected to amplify target insecticidalprotein genes that encode a protein with at least 50% sequence identityto SEQ ID NO: 87.

In some embodiments, the disclosure provides for an isolated nucleicacid molecule encoding an insecticidal protein having at least about80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to a proteinselected from the group consisting of: SEQ ID NO: 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, and 72. In certainembodiments, the isolated nucleic acid molecule is codon optimized forexpression in a host cell of interest. In certain embodiments, theisolated nucleic acid molecule is codon optimized for expression in aplant cell. In certain embodiments, the isolated nucleic acid moleculehas at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequenceidentity to a nucleic acid sequence selected from the group consistingof: SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65,67, 69, and 71.

In some embodiments, the disclosure provides for a nucleotide construct,comprising: a nucleic acid molecule encoding an insecticidal proteinhaving at least about 80% sequence identity to a protein selected fromthe group consisting of: SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,58, 60, 62, 64, 66, 68, 70, and 72, said nucleic acid molecule operablylinked to a heterologous regulatory element. In aspects, theheterologous regulatory element is a promoter. In aspects, theheterologous regulatory element is a plant promoter. In someembodiments, the disclosure provides for transgenic plant cells thatcomprise said nucleotide constructs. In some embodiments, the disclosureprovides for stably transformed plants that express said proteins fromthe nucleotide construct. In some embodiments, insects feed upon thetransgenic plants and are killed.

In some embodiments, the disclosure provides for an isolatedinsecticidal protein, comprising: an amino acid sequence with at leastabout 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to anamino acid sequence selected from the group consisting of: SEQ ID NO: 2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, and 72. Insome embodiments, the isolated insecticidal protein is recombinant. Insome embodiments, the disclosure provides for transgenic plant cellsthat express said proteins. In some embodiments, insects feed upon thetransgenic plants and are killed. In some embodiments, theaforementioned insecticidal proteins are contained in agriculturalcompositions. In some embodiments, said agricultural compositions areused to spray upon plants and/or insects, in order to provide effectiveinsect control. In some embodiments, the insecticidal proteins are foundin cell lysate and the cell lysate can be utilized to control insectpest populations. In some embodiments, the natural Pseudomonas hostorganism can be formulated into a composition and utilized to combatinsect pests.

In certain embodiments, the disclosure provides novel insecticidalproteins, wherein the proteins having an amino acid sequence which scoreat or above a bit score of 521.5 and/or sequences which match at anE-value of less than or equal to 7.9e-161 when scored using the HMM inTable 6. These proteins can be provided in any form (e.g., as isolatedor recombinant proteins) or as part of any of the compositions (e.g.,plants or agricultural compositions) disclosed herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 outlines a workflow of the taught insecticidal protein discoveryplatform (IPDP).

FIG. 2 outlines a workflow of the taught insecticidal protein discoveryplatform (IPDP) and illustrates two steps utilized by methods of theprior art, which are not required by the current IPDP.

FIG. 3 illustrates a multiple sequence alignment of eight novelinsecticidal proteins (ZIP1, ZIP2, ZIP6, ZIP8, ZIP9, ZIP10, ZIP11,ZIP12) found in Table 3, which were discovered utilizing the IPDP.

FIG. 4 illustrates a multiple sequence alignment of eight novelinsecticidal proteins (ZIP1, ZIP2, ZIP6, ZIP8, ZIP9, ZIP10, ZIP11,ZIP12) found in Table 3, which were discovered utilizing the IPDP, ascompared to monalysin.

FIG. 5 illustrates a phylogenetic tree of eight novel insecticidalproteins found in Table 3 and FIG. 3, which were discovered utilizingthe IPDP.

FIG. 6 illustrates a phylogenetic tree of eight novel insecticidalproteins found in Table 3 and FIG. 4, which were discovered utilizingthe IPDP, as compared to monalysin.

FIG. 7 illustrates the results of insect bioassay experiments with tenpurified insecticidal proteins found in Table 3. Insects (Halyomorphahalys—Brown Marmorated Stink Bug) that ingested water containingpurified insecticidal proteins (ZIP1, ZIP2, ZIP4, ZIP6, ZIP8, ZIP9,ZIP10, ZIP11, and ZIP12, and ZIP16) discovered via the IPDP exhibitedmortality rates of varying degrees. The concentration of purifiedinsecticidal protein used for this experiment is also presented in FIG.7.

FIG. 8 illustrates the results of insect bioassay experiments with threepurified insecticidal proteins (ZIP1, ZIP2, and ZIP4) found in Table 3against Brown Marmorated Stinkbugs. Purified proteins of varyingconcentrations were ingested by insects (N=number of insects assayed)and the mortality data was subject to Probit Analysis to generate thelethal concentration required to kill 50% of the population (LC50) withupper and lower 95% confidence intervals.

FIGS. 9A-B illustrates the results of insect bioassay experiments withthree purified insecticidal proteins (ZIP1, ZIP2, and ZIP4) found inTable 3 against members of two major Orders of insects; Fall Armywormand Southern Corn Rootworm. The percent reduction in the mean weight ofinsects that ingested the listed concentration of purified protein mixedwith solid diet as compared to buffer only control is reported. FIG. 9Apresents experiments performed on Fall Armyworm (Spodoptera frugiperda),while FIG. 9B illustrates experiments performed on Southern CornRootworm (Diabrotica undecimpunctata)

FIG. 10 illustrates the results from an insect lysate experiment.Insects (Halyomorpha halys—Brown Marmorated Stink Bug) that ingestedbacterial lysate containing an insecticidal protein discovered via theIPDP exhibited a 100% mortality rate.

FIG. 11 illustrates the western blot results from Example 6, which showsexpression of ZIP proteins from soybean and corn leaves. Lanes 1 and 11:Negative Control (untransformed soybean leaves); Lanes 2 and 3: ZIP1Transformed soybean leaves; Lanes 4-10: ZIP2 Transformed soybean leaves;Lanes 12 and 13: ZIP4 Transformed soybean leaves; Lane 14: NegativeControl (untransformed maize leaves); Lanes 15 and 16: ZIP2 Transformedmaize leaves.

DETAILED DESCRIPTION Definitions

While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the presently disclosed subject matter.

The term “a” or “an” refers to one or more of that entity, i.e. canrefer to a plural referents. As such, the terms “a” or “an”, “one ormore” and “at least one” are used interchangeably herein. In addition,reference to “an element” by the indefinite article “a” or “an” does notexclude the possibility that more than one of the elements is present,unless the context clearly requires that there is one and only one ofthe elements.

As used herein the terms “cellular organism” “microorganism” or“microbe” should be taken broadly. These terms are used interchangeablyand include, but are not limited to, the two prokaryotic domains,Bacteria and Archaea, as well as certain eukaryotic fungi and protists.In some embodiments, the disclosure refers to the “microorganisms” or“cellular organisms” or “microbes” of lists/tables and figures presentin the disclosure. This characterization can refer to not only theidentified taxonomic genera of the tables and figures, but also theidentified taxonomic species, as well as the various novel and newlyidentified or designed strains of any organism in said tables orfigures. The same characterization holds true for the recitation ofthese terms in other parts of the Specification, such as in theExamples.

The term “prokaryotes” is art recognized and refers to cells whichcontain no nucleus or other cell organelles. The prokaryotes aregenerally classified in one of two domains, the Bacteria and theArchaea. The definitive difference between organisms of the Archaea andBacteria domains is based on fundamental differences in the nucleotidebase sequence in the 16S ribosomal RNA.

The term “Archaea” refers to a categorization of organisms of thedivision Mendosicutes, typically found in unusual environments anddistinguished from the rest of the prokaryotes by several criteria,including the number of ribosomal proteins and the lack of muramic acidin cell walls. On the basis of ssrRNA analysis, the Archaea consist oftwo phylogenetically-distinct groups: Crenarchaeota and Euryarchaeota.On the basis of their physiology, the Archaea can be organized intothree types: methanogens (prokaryotes that produce methane); extremehalophiles (prokaryotes that live at very high concentrations of salt(NaCl); and extreme (hyper) thermophilus (prokaryotes that live at veryhigh temperatures). Besides the unifying archaeal features thatdistinguish them from Bacteria (i.e., no murein in cell wall,ester-linked membrane lipids, etc.), these prokaryotes exhibit uniquestructural or biochemical attributes which adapt them to theirparticular habitats. The Crenarchaeota consists mainly ofhyperthermophilic sulfur-dependent prokaryotes and the Euryarchaeotacontains the methanogens and extreme halophiles.

“Bacteria” or “eubacteria” refers to a domain of prokaryotic organisms.Bacteria include at least 11 distinct groups as follows: (1)Gram-positive (gram+) bacteria, of which there are two majorsubdivisions: (1) high G+C group (Actinomycetes, Mycobacteria,Micrococcus, others) (2) low G+C group (Bacillus, Clostridia,Lactobacillus, Staphylococci, Streptococci, Mycoplasmas); (2)Proteobacteria, e.g., Purple photosynthetic+non-photosyntheticGram-negative bacteria (includes most “common” Gram-negative bacteria);(3) Cyanobacteria, e.g., oxygenic phototrophs; (4) Spirochetes andrelated species; (5) Planctomyces; (6) Bacteroides, Flavobacteria; (7)Chlamydia; (8) Green sulfur bacteria; (9) Green non-sulfur bacteria(also anaerobic phototrophs); (10) Radioresistant micrococci andrelatives; (11) Thermotoga and Thermosipho thermophiles.

A “eukaryote” is any organism whose cells contain a nucleus and otherorganelles enclosed within membranes. Eukaryotes belong to the taxonEukarya or Eukaryota. The defining feature that sets eukaryotic cellsapart from prokaryotic cells (the aforementioned Bacteria and Archaea)is that they have membrane-bound organelles, especially the nucleus,which contains the genetic material, and is enclosed by the nuclearenvelope.

The terms “genetically modified host cell,” “recombinant host cell,” and“recombinant strain” are used interchangeably herein and refer to hostcells that have been genetically modified by the cloning andtransformation methods of the present disclosure. Thus, the termsinclude a host cell (e.g., bacteria, yeast cell, fungal cell, CHO, humancell, etc.) that has been genetically altered, modified, or engineered,such that it exhibits an altered, modified, or different genotype and/orphenotype (e.g., when the genetic modification affects coding nucleicacid sequences of the microorganism), as compared to thenaturally-occurring organism from which it was derived. It is understoodthat in some embodiments, the terms refer not only to the particularrecombinant host cell in question, but also to the progeny or potentialprogeny of such a host cell.

The term “wild-type microorganism” or “wild-type host cell” describes acell that occurs in nature, i.e. a cell that has not been geneticallymodified.

The term “genetically engineered” may refer to any manipulation of ahost cell's genome (e.g. by insertion, deletion, mutation, orreplacement of nucleic acids).

The term “control” or “control host cell” refers to an appropriatecomparator host cell for determining the effect of a geneticmodification or experimental treatment. In some embodiments, the controlhost cell is a wild type cell. In other embodiments, a control host cellis genetically identical to the genetically modified host cell, save forthe genetic modification(s) differentiating the treatment host cell.

As used herein, the term “allele(s)” means any of one or morealternative forms of a gene, all of which alleles relate to at least onetrait or characteristic. In a diploid cell, the two alleles of a givengene occupy corresponding loci on a pair of homologous chromosomes.

As used herein, the term “locus” (loci plural) means a specific place orplaces or a site on a chromosome where for example a gene or geneticmarker is found.

As used herein, the term “genetically linked” refers to two or moretraits that are co-inherited at a high rate during breeding such thatthey are difficult to separate through crossing.

A “recombination” or “recombination event” as used herein refers to achromosomal crossing over or independent assortment.

As used herein, the term “phenotype” refers to the observablecharacteristics of an individual cell, cell culture, organism, or groupof organisms which results from the interaction between thatindividual's genetic makeup (i.e., genotype) and the environment.

As used herein, the term “chimeric” or “recombinant” when describing anucleic acid sequence or a protein sequence refers to a nucleic acid, ora protein sequence, that links at least two heterologouspolynucleotides, or two heterologous polypeptides, into a singlemacromolecule, or that rearranges one or more elements of at least onenatural nucleic acid or protein sequence. For example, the term“recombinant” can refer to an artificial combination of two otherwiseseparated segments of sequence, e.g., by chemical synthesis or by themanipulation of isolated segments of nucleic acids by geneticengineering techniques.

As used herein, a “synthetic nucleotide sequence” or “syntheticpolynucleotide sequence” is a nucleotide sequence that is not known tooccur in nature or that is not naturally occurring. Generally, such asynthetic nucleotide sequence will comprise at least one nucleotidedifference when compared to any other naturally occurring nucleotidesequence.

As used herein, a “synthetic amino acid sequence” or “synthetic peptide”or “synthetic protein” is an amino acid sequence that is not known tooccur in nature or that is not naturally occurring. Generally, such asynthetic protein sequence will comprise at least one amino aciddifference when compared to any other naturally occurring proteinsequence.

As used herein, the term “nucleic acid” refers to a polymeric form ofnucleotides of any length, either ribonucleotides ordeoxyribonucleotides, or analogs thereof. This term refers to theprimary structure of the molecule, and thus includes double- andsingle-stranded DNA, as well as double- and single-stranded RNA. It alsoincludes modified nucleic acids such as methylated and/or capped nucleicacids, nucleic acids containing modified bases, backbone modifications,and the like. The terms “nucleic acid” and “nucleotide sequence” areused interchangeably.

As used herein, the term “gene” refers to any segment of DNA associatedwith a biological function. Thus, genes include, but are not limited to,coding sequences and/or the regulatory sequences required for theirexpression. Genes can also include non-expressed DNA segments that, forexample, form recognition sequences for other proteins. Genes can beobtained from a variety of sources, including cloning from a source ofinterest or synthesizing from known or predicted sequence information,and may include sequences designed to have desired parameters.

As used herein, the term “homologous” or “homologue” or “ortholog” isknown in the art and refers to related sequences that share a commonancestor or family member and can be inferred based on the degree ofsequence identity. The terms “homology,” “homologous,” “substantiallysimilar” and “corresponding substantially” are used interchangeablyherein. They refer to nucleic acid fragments wherein changes in one ormore nucleotide bases do not affect the ability of the nucleic acidfragment to mediate gene expression or produce a certain phenotype.These terms also refer to modifications of the nucleic acid fragments ofthe instant disclosure such as deletion or insertion of one or morenucleotides that do not substantially alter the functional properties ofthe resulting nucleic acid fragment relative to the initial, unmodifiedfragment. It is therefore understood, as those skilled in the art willappreciate, that the disclosure encompasses more than the specificexemplary sequences. These terms describe the relationship between agene found in one species, subspecies, variety, cultivar or strain andthe corresponding or equivalent gene in another species, subspecies,variety, cultivar or strain. For purposes of this disclosure homologoussequences are compared. “Homologous sequences” or “homologues” or“orthologs” are thought, believed, or known to be functionally related.A functional relationship may be indicated in any one of a number ofways, including, but not limited to: (a) degree of sequence identityand/or (b) the same or similar biological function. Preferably, both (a)and (b) are indicated. Homology can be determined using softwareprograms readily available in the art, such as those discussed inCurrent Protocols in Molecular Biology (F. M. Ausubel et al., eds.,1987) Supplement 30, section 7.718, Table 7.71. Some alignment programsare MacVector (Oxford Molecular Ltd, Oxford, U.K.), ALIGN Plus(Scientific and Educational Software, Pennsylvania) and AlignX (VectorNTI, Invitrogen, Carlsbad, Calif.). Another alignment program isSequencher (Gene Codes, Ann Arbor, Mich.), using default parameters.

As used herein, the term “endogenous” or “endogenous gene,” refers tothe naturally occurring gene, in the location in which it is naturallyfound within the host cell genome. In the context of the presentdisclosure, operably linking a heterologous promoter to an endogenousgene means genetically inserting a heterologous promoter sequence infront of an existing gene, in the location where that gene is naturallypresent. An endogenous gene as described herein can include alleles ofnaturally occurring genes that have been mutated according to any of themethods of the present disclosure.

As used herein, the term “exogenous” is used interchangeably with theterm “heterologous,” and refers to a substance coming from some sourceother than its native source. For example, the terms “exogenousprotein,” or “exogenous gene” refer to a protein or gene from anon-native source or location, and that have been artificially suppliedto a biological system.

As used herein, the term “nucleotide change” refers to, e.g., nucleotidesubstitution, deletion, and/or insertion, as is well understood in theart. For example, mutations contain alterations that produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded protein or how the proteins are made.

As used herein, the term “protein modification” refers to, e.g., aminoacid substitution, amino acid modification, deletion, and/or insertion,as is well understood in the art.

As used herein, the term “at least a portion” or “fragment” of a nucleicacid or polypeptide means a portion having the minimal sizecharacteristics of such sequences, or any larger fragment of the fulllength molecule, up to and including the full length molecule. Afragment of a polynucleotide of the disclosure may encode a biologicallyactive portion of a genetic regulatory element. A biologically activeportion of a genetic regulatory element can be prepared by isolating aportion of one of the polynucleotides of the disclosure that comprisesthe genetic regulatory element and assessing activity as describedherein. Similarly, a portion of a polypeptide may be 4 amino acids, 5amino acids, 6 amino acids, 7 amino acids, and so on, going up to thefull length polypeptide. The length of the portion to be used willdepend on the particular application. A portion of a nucleic acid usefulas a hybridization probe may be as short as 12 nucleotides; in someembodiments, it is 20 nucleotides. A portion of a polypeptide useful asan epitope may be as short as 4 amino acids. A portion of a polypeptidethat performs the function of the full-length polypeptide wouldgenerally be longer than 4 amino acids.

Variant polynucleotides also encompass sequences derived from amutagenic and recombinogenic procedure such as DNA shuffling. Strategiesfor such DNA shuffling are known in the art. See, for example, Stemmer(1994) PNAS 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameriet al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol.Biol. 272:336-347; Zhang et al. (1997) PNAS 94:4504-4509; Crameri et al.(1998) Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

For PCR amplifications of the polynucleotides disclosed herein,oligonucleotide primers can be designed for use in PCR reactions toamplify corresponding DNA sequences from cDNA or genomic DNA extractedfrom any organism of interest. Methods for designing PCR primers and PCRcloning are generally known in the art and are disclosed in Sambrook etal. (2001) Molecular Cloning: A Laboratory Manual (3^(rd) ed., ColdSpring Harbor Laboratory Press, Plainview, New York). See also Innis etal., eds. (1990) PCR Protocols: A Guide to Methods and Applications(Academic Press, New York); Innis and Gelfand, eds. (1995) PCRStrategies (Academic Press, New York); and Innis and Gelfand, eds.(1999) PCR Methods Manual (Academic Press, New York). Known methods ofPCR include, but are not limited to, methods using paired primers,nested primers, single specific primers, degenerate primers,gene-specific primers, vector-specific primers, partially-mismatchedprimers, and the like.

The term “primer” as used herein refers to an oligonucleotide which iscapable of annealing to the amplification target allowing a DNApolymerase to attach, thereby serving as a point of initiation of DNAsynthesis when placed under conditions in which synthesis of primerextension product is induced, i.e., in the presence of nucleotides andan agent for polymerization such as DNA polymerase and at a suitabletemperature and pH. The (amplification) primer is preferably singlestranded for maximum efficiency in amplification. Preferably, the primeris an oligodeoxyribonucleotide. The primer must be sufficiently long toprime the synthesis of extension products in the presence of the agentfor polymerization. The exact lengths of the primers will depend on manyfactors, including temperature and composition (A/T vs. G/C content) ofprimer. A pair of bi-directional primers consists of one forward and onereverse primer as commonly used in the art of DNA amplification such asin PCR amplification.

As used herein, “promoter” refers to a DNA sequence capable ofcontrolling the expression of a coding sequence or functional RNA. Insome embodiments, the promoter sequence consists of proximal and moredistal upstream elements, the latter elements often referred to asenhancers. Accordingly, an “enhancer” is a DNA sequence that canstimulate promoter activity, and may be an innate element of thepromoter or a heterologous element inserted to enhance the level ortissue specificity of a promoter. Promoters may be derived in theirentirety from a native gene, or be composed of different elementsderived from different promoters found in nature, or even comprisesynthetic DNA segments. It is understood by those skilled in the artthat different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental conditions. It is furtherrecognized that since in most cases the exact boundaries of regulatorysequences have not been completely defined, DNA fragments of somevariation may have identical promoter activity.

As used herein, the phrases “recombinant construct”, “expressionconstruct”, “chimeric construct”, “construct”, and “recombinant DNAconstruct” are used interchangeably herein. A recombinant constructcomprises an artificial combination of nucleic acid fragments, e.g.,regulatory and coding sequences that are not found together in nature.For example, a chimeric construct may comprise regulatory sequences andcoding sequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different from that found in nature. Such constructmay be used by itself or may be used in conjunction with a vector. If avector is used then the choice of vector is dependent upon the methodthat will be used to transform host cells as is well known to thoseskilled in the art. For example, a plasmid vector can be used. Theskilled artisan is well aware of the genetic elements that must bepresent on the vector in order to successfully transform, select andpropagate host cells comprising any of the isolated nucleic acidfragments of the disclosure. The skilled artisan will also recognizethat different independent transformation events will result indifferent levels and patterns of expression (Jones et al., (1985) EMBOJ. 4:2411-2418; De Almeida et al., (1989) Mol. Gen. Genetics 218:78-86),and thus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA, Northern analysis of mRNAexpression, immunoblotting analysis of protein expression, or phenotypicanalysis, among others. Vectors can be plasmids, viruses,bacteriophages, pro-viruses, phagemids, transposons, artificialchromosomes, and the like, that replicate autonomously or can integrateinto a chromosome of a host cell. A vector can also be a naked RNApolynucleotide, a naked DNA polynucleotide, a polynucleotide composed ofboth DNA and RNA within the same strand, a poly-lysine-conjugated DNA orRNA, a peptide-conjugated DNA or RNA, a liposome-conjugated DNA, or thelike, that is not autonomously replicating. As used herein, the term“expression” refers to the production of a functional end-product e.g.,an mRNA or a protein (precursor or mature).

“Operably linked” means in this context the sequential arrangement ofthe promoter polynucleotide according to the disclosure with a furtheroligo- or polynucleotide, resulting in transcription of said furtherpolynucleotide.

The term “product of interest” or “biomolecule” as used herein refers toany product produced by microbes from feedstock. In some cases, theproduct of interest may be a small molecule, enzyme, peptide, aminoacid, organic acid, synthetic compound, fuel, alcohol, etc. For example,the product of interest or biomolecule may be any primary or secondaryextracellular metabolite. The primary metabolite may be, inter alia,ethanol, citric acid, lactic acid, glutamic acid, glutamate, lysine,threonine, tryptophan and other amino acids, vitamins, polysaccharides,etc. The secondary metabolite may be, inter alia, an antibiotic compoundlike penicillin, or an immunosuppressant like cyclosporin A, a planthormone like gibberellin, a statin drug like lovastatin, a fungicidelike griseofulvin, etc. The product of interest or biomolecule may alsobe any intracellular component produced by a microbe, such as: amicrobial enzyme, including: catalase, amylase, protease, pectinase,glucose isomerase, cellulase, hemicellulase, lipase, lactase,streptokinase, and many others. The intracellular component may alsoinclude recombinant proteins, such as: insulin, hepatitis B vaccine,interferon, granulocyte colony-stimulating factor, streptokinase andothers.

The term “carbon source” generally refers to a substance suitable to beused as a source of carbon for cell growth. Carbon sources include, butare not limited to, biomass hydrolysates, starch, sucrose, cellulose,hemicellulose, xylose, and lignin, as well as monomeric components ofthese substrates. Carbon sources can comprise various organic compoundsin various forms, including, but not limited to polymers, carbohydrates,acids, alcohols, aldehydes, ketones, amino acids, peptides, etc. Theseinclude, for example, various monosaccharides such as glucose, dextrose(D-glucose), maltose, oligosaccharides, polysaccharides, saturated orunsaturated fatty acids, succinate, lactate, acetate, ethanol, etc., ormixtures thereof. Photosynthetic organisms can additionally produce acarbon source as a product of photosynthesis. In some embodiments,carbon sources may be selected from biomass hydrolysates and glucose.

The term “feedstock” is defined as a raw material or mixture of rawmaterials supplied to a microorganism or fermentation process from whichother products can be made. For example, a carbon source, such asbiomass or the carbon compounds derived from biomass are a feedstock fora microorganism that produces a product of interest (e.g. smallmolecule, peptide, synthetic compound, fuel, alcohol, etc.) in afermentation process. However, a feedstock may contain nutrients otherthan a carbon source.

The term “volumetric productivity” or “production rate” is defined asthe amount of product formed per volume of medium per unit of time.Volumetric productivity can be reported in gram per liter per hour(g/L/h).

The term “specific productivity” is defined as the rate of formation ofthe product. Specific productivity is herein further defined as thespecific productivity in gram product per gram of cell dry weight (CDW)per hour (g/g CDW/h). Using the relation of CDW to OD₆₀₀ for the givenmicroorganism specific productivity can also be expressed as gramproduct per liter culture medium per optical density of the culturebroth at 600 nm (OD) per hour (g/L/h/OD).

The term “yield” is defined as the amount of product obtained per unitweight of raw material and may be expressed as g product per g substrate(g/g). Yield may be expressed as a percentage of the theoretical yield.“Theoretical yield” is defined as the maximum amount of product that canbe generated per a given amount of substrate as dictated by thestoichiometry of the metabolic pathway used to make the product.

The term “titre” or “titer” is defined as the strength of a solution orthe concentration of a substance in solution. For example, the titre ofa product of interest (e.g. small molecule, peptide, synthetic compound,fuel, alcohol, etc.) in a fermentation broth is described as g ofproduct of interest in solution per liter of fermentation broth (g/L).

The term “total titer” is defined as the sum of all product of interestproduced in a process, including but not limited to the product ofinterest in solution, the product of interest in gas phase ifapplicable, and any product of interest removed from the process andrecovered relative to the initial volume in the process or the operatingvolume in the process.

The term “insecticidal protein” or “pesticidal protein” or “insecticidaltoxin” or “pesticidal toxin” is used to refer to a protein that hastoxic activity against one or more pests. Examples of pests includevarious orders of insects, including: Lepidopterans, Dipterans,Hemipterans, and Coleopterans, to name a few. Pests also includenon-insect organisms that are a pest to agriculture, including forexample, members of the Nematoda phylum.

Insecticidal/Pesticidal Proteins

The disclosure teaches an insecticidal protein discovery platform andinsecticidal proteins discovered therefrom. However, it should beunderstood that the term “insecticidal” is not limited to merelyinsects, but rather covers a broader taxonomic grouping of organismsthat are commonly referred to as “pests.” Consequently, the phrase“insecticidal protein” can be taken to be synonymous with “pesticidalprotein” and the phrase “insecticidal protein discovery platform” can betaken to be synonymous with “pesticidal protein discovery platform.”Furthermore, in some aspects, the disclosure provides for insecticidaltoxins and a platform for discovering insecticidal toxins, which may notbe limited to protein embodiments.

Insecticidal Proteins—Monalysins

Pseudomonas entomophila is an entomopathogenic bacterium that infectsand kills Drosophila. P. entomophila pathogenicity is linked to itsability to cause irreversible damages to the Drosophila gut, preventingepithelium renewal and repair. Recently, Opota and colleagues reportedthe identification of a novel pore-forming toxin (PFT), which theytermed “Monalysin,” contributes to the virulence of P. entomophilaagainst Drosophila. Opota, et al., “Monalysin, a Novel B-Pore-FormingToxin from the Drosophila Pathogen Pseudomonas entomophila, Contributesto Host Intestinal Damage and Lethality,” PLoS Pathogens, September2011, Vol. 7, Issue 9. Opota demonstrated Monalysin requires N-terminalcleavage to become fully active, forms oligomers in vitro, and inducespore-formation in artificial lipid membranes. The prediction of thesecondary structure of the membrane-spanning domain indicates thatMonalysin is a PFT of the B-type. The expression of Monalysin isregulated by both the GacS/GacA two-component system and the Pvfregulator, two signaling systems that control P. entomophilapathogenicity. In addition, AprA, a metallo-protease secreted by P.entomophila, can induce the rapid cleavage of pro-Monalysin into itsactive form. Reduced cell death is observed upon infection with a mutantdeficient in Monalysin production showing that Monalysin plays a role inP. entomophila ability to induce intestinal cell damages, which isconsistent with its activity as a PFT. Opota's study, together with thewell-established action of Bacillus thuringiensis Cry toxins, suggeststhat production of PFTs is a common strategy of entomopathogens todisrupt insect gut homeostasis. Id.

Opota discovered Monalysin (PSEEN3174), by characterizing the proteinproduct of the unknown gene pseen3174. According to Opota, the Monalysinamino acid sequence does not show homology to other sequences using PBlast, except for two uncharacterized orthologs found in Pseudomonasputida F1 strain (FIG. S1 of Opota). Neither the P. entomophila nor theP. putida gene products displayed any obvious protein domains. However,Opota utilized the HHpred software (Homology detection & structureprediction by HMM-HMM comparison) to reveal the presence of an internalregion with alternating polar and hydrophobic residues flanked by astretch of serine and threonine residues, a hallmark of themembrane-spanning region of B-barrel pore-forming toxins. Id.

Opota's DNA sequence searches and analysis were performed using thePseudomonas genome database (pseudomonas.com, which can be accessed onthe worldwide web using the “www” prefix). The monalysin gene (ORFPSEEN3174) corresponds to the accession number YP_608728.1. Monalysinputative orthologs in Pseudomonas putida Pput_1063 and Pput_1064correspond to the accessions numbers YP_001266408.1, YP_001266409.1respectively. The ORF PSEEN0535 involved in the production of the typeVI secretion system corresponds to the accession number YP_606298.1.

Insecticidal Proteins—Pseudomonas Insecticidal Proteins (PIPs)

There are several known families of Pseudomonas insecticidal proteins,including: PIP-1, 45, 47, 64, 72, 74, 75, and 77. These PIP proteins,along with identifying characteristics, are provided in the belowTable 1. Further information can be found in: (1) U. Schellenberger etal., “A selective insecticidal protein from Pseudomonas for controllingcorn rootworms,” Science, 2016 Nov. 4; 354(6312):634-637 (providingIPD072Aa, an 86 AA protein, GenBank Accession No. KT795291) incorporatedby reference herein; and (2) Jun-Zhi Wei et al., “A selectiveinsecticidal protein from Pseudomonas mosselii for corn rootwormcontrol,” Plant Biotechnology Journal, 2018, Vol. 16, pgs. 649-659(providing PIP-47aa) incorporated by reference herein.

TABLE 1 Pseudomonas insecticidal proteins (PIPs) and Monalysin PIPSource Source Publication¹ Identifier Amino Acid Sequence² Organism13792861/ PIP-1 SEQ ID NO: 2 P. chlororaphis US9688730B2MPIKEELSQPQSHSIELDDLKSEQGSLRAAL TSNFAGNFDQFPTKRGGFAIDSYLLDYSAPKQGCWVDGITVYGDIFIGKQNWGTYTRPVFAY LQYMDTISIPQQVTQTRSYQLTKGHTKTFITNVSAKYSVGGSIDIVNVGSDISIGFSNSESW STTQTFSNSTQLTGPGTFIVYQVVMVYAHNATSAGRQNGNAFAYNKTNTVGSRLDLYYLSAI TQNSTVIVDSSKAIAPLDWDTVQRNVLMENYNPGSNSGHFSFDWSAYNDPHRRY (SEQ ID NO: 73) 15543689/ PIP-45-1 SEQ ID NO: 1P. brenneri US20170367349A1 MSTPFKQFTSPAGQAPKDYNKLGLENQLPQFETDWNNDLTGWTQSAIIGNPWSGLNDAPRSG YYNPLVEGYGPTTPPAITWAPFPNRLWTFFYNNGTAVIPQLGGKAMSLQQVMELTDNGQITI NNTLYMLYDPNKQGTLLQLPVTRCPTIDWQGKYKDFSPSGPRGWLDEYCEWSIVRDADGNMR KITFTCENPAYFLAMWRIDPNAVLGLYRDYIDPQVQLEDLYLRYTADCPTGKAGDPVIDPTT GQPAYDTVNKWNAGTACVPGQYGGAMHLTSGPNTLSAEVYLAAAATILRPLASSQNSQALIC CAQYGQNYRNSDPHIGFSANSVAVNNRLSLTNPIGLYLQQPTDFSAWKGPQGQDVSQYWKIT RGTAKSAANGSDQILQAVFEVPVSAGFSINDITISGQPIDYVWVIAQQLLVGLSVTTTPISP TPDSCPCVKDRVNGVQPWPVQLLPLDLFYGQSPTDLPAWLAPGTSGQFALVVQGADLKTTAE TARVQFSNPGVTAQVTQFLPDASAIPGQTNSGGTQGYLLTITVSPTAAPGLVTVRALNPGEA DNPSATEHPWESGLALVPGA (SEQ ID NO: 74)15543689/ PIP-45-2 SEQ ID NO: 2 P. brenneri US20170367349A1MSRLRLSVLSLLTSVVLSLFAMQAAYASPTS DADACVQQQLVFNPKSGGFLPINNFNATGQSFMNCFGWQLFIALNWPVNPGWPATPALAGEP DMNSTLAQFGVPTASGQPMSVAPVWASYKDANDIFLPGAPAPTGWGVQTLVPSNCSTQGSLR AISVGARKFMTATSESAINARHGFHLSSGTLASIPDPIMEASGGWLTDQSQNLVFFERKVGK AEFDYIVSKGLYDAANQLTVAQNLDNQNPGGLSLPIGEPMRSLPPNPVPQEQLGALEVKAAW RILTGKPELYGRYLTTVAWLKNPATLQCTQQVVGLVGLHIINKTQASPNFIWTTFEQVDNVP EPNQVPPQQTPPDSFAFNNPNCGTGPECTPNVARIQCKQHHPDRDCTEPFPRDQPVQTTREH PLPTELQALNGAVQANFAQQSQGKSVFQYYKLINVLWTLTPNPPTQPEPGVSAQVPLSYGPF ISQGNVPVANTTLETYVQGDNCNACHQYATIAGSSTLASDFSFLFNSADSASKNSLVKRVKA FQTLKDQP (SEQ ID NO: 75) 15543689/PIP-64-1 SEQ ID NO: 53 P. brenneri US20170367349A1MGSITDHNQLLAWVASLDIPEASGVKTRSRN VVARANAEDEGAAVVRGSITSFVTGLSQQARDDVQNSTLLMQLAADKKFNPEKQREEWFKFY TDGLANLGWGRVSSYYQSYQPRNTNVTMDQVVLEVIAAVVGADSAVYKVTEKTFSSLQDNPK NQAPLKLFDSSSTRDSVGTFQILPVMQDRDGNVVMVLTTVNASTTVQRGSFLFWSWSKTTAW MYRAAQQTVLNESVYATVRQSVIKKLGKNAE EFIDDLEI(SEQ ID NO: 76) SEQ ID NO: 54 15543689/ PIP-64-2MKLSADEVYVISGNLLSATPSLTDPTVLEDI P. brenneri US20170367349A1ANSNLLCQLAADKNQGTRFIDPAAWLDFYRS SLGRLFWRISNSGTVSYAIPQLVHKITVKEVLEKTFYKTLDRPQRIRVEESIELLGEQSADS PSATLYSLKTQVNFNETTSSPGLLPHSISSVNLQLSVVHSETCISVCSVYFKTSTRIGDDVF NQKFPVKELLGNVSVSTFEAKLLESSYAGIRQSIIDKLGEDNIRENILLVPAVSPSLSNTRH AGALQFVQELDI (SEQ ID NO: 77)SEQ ID NO: 73 15543689/ PIP-74-1 MAKLTQFSTPADIQDFSDSPAQQERMNAAWSP. rhodesiae US20170367349A1 GNINRWVNAALVGDVWDLINYGPRPAFYNPLDTDTPSTSVNAPITWNAFPGRIPALFPNQSA NWLQWADQGVPANVTTNLCTQQSVPPAPYSPTGPRGWQDEYCEWSVTRNAAGQITSVMFTCE NPEYWMTLWQVDPGKVLQRYQQLINPAVQLADLSLKDAQGQTVIDPVTGAPCYNPLNKWNSG TQTLPGSGGAMHLTSSPNTLGAEYDLAAAATMPRELNNEPVTSASQLVCYARYGRIGRHSDP TIGQNVNQYVNYTSGLTEVRATLTNPPGLYIQTPDFSGYTTPDGSPAAACWTINRGHLAQTS DDIDRILHATFSVPAGKNFTVSDISINGAKIQYASQIAGTITMGLMATVFGNSGVTQQPVAG TLDSDNPSPSVSALQPLSVFNAYRAQELASNEQALSIPILALAIRPGQQVDNIALLLNTSQT PNGASFSVVEGGVSISITGTQDLPGLDMSLYLVSISADANAAPGDRTVLASVPGMASTQQAA IGLLTVGGPTLVTSQTGPSKPNFRRGRG(SEQ ID NO: 78) 15543689/ PIP-74-2 SEQ ID NO: 74 P. rhodesiaeUS20170367349A1 MRRRPTVLLGLALLLGLPATQAMGAPLCGSPFVPSPTLQPTLAPPNFSASDSAVDCFMWQTM VYLNWPATPGQRGVPNAAASLGSPGPSVWQTYKDYNELYLPNGQQPPAWNDNFLSVQRLQTR GVARALPSIRLLNSTSKVFRAANANESPALREIEQVGGGVLYDQAGSPVYYEMLVNEVNFDF IYNNQLYNPAQQNLYAKQKGIVLPNNSIEIKAAWKVLSDPDNPQRFLTAQALLPGSSTPVTV GLVGLHVFQMPSSAFNQGFWATFQQLDNAPTVAGATPGAHYSFNNPQCAPAQCPPNDKTSNP TQVVQNFPPTPEAQNINHYMQNLIAQQAPGSALQYYQLVDVQWPTSPQAIGQPGATAPAPSG TPNHDTLINPVLETFLQANHKSCLGCHVYASVAADGSNPPTHYQASFSFLLGHAKSPALGSN LKSLAQQIEDASLSLQH (SEQ ID NO: 79)15543689/ PIP-75 SEQ ID NO: 79 P. antarctica US20170367349A1MKLSNVLLLSIVFAWQGMAFADTQKSNAETL LSNDKPPLTQAAQEKEQENVEADRNECWSAKNCSGKILNNKDAHNCKLSGGKSWRSKTTGQC TNL (SEQ ID NO: 80) 15543689/ PIP-77SEQ ID NO: 88 P. chlororaphis US20170367349A1MSAQENFVGGWTPYHKLTPKDQEVFKEALAG FVGVQYTPELVSTQVVNGTNYRYQSKATLPGSSESWQAVVEIYAPIKGKPHITQIHRI (SEQ ID NO: 81) 14912356/ PIP-47AaSEQ ID NO: 2 P. putida US20160186204A1 MHAPGAIPSEKESAHAWLTETKANAKSTALRGNIFAQDYNRQLLTATGQSMRSGADAINPFF SPAKGTATGSYAKDADANVSPGSAPVSIYEGLQTAIDIARRRSGYNPLDQPTDQKPKSAGDR EHFIAFTQQIAEIPFLSLLAAQVTQIQQKSHDANALVDSFVKGFIGLKNQDVEQIKQSLSSL VNAALSYSEQTERQSNFNQNILQTGDSGSVNFMLYASEFTIKASSHKGTITFQSSYTLSQAI YQLSVESWNNVKDVFSKQQKTDTQQWLGDTTTQVREGSKLRAICLVS (SEQ ID NO: 82) 14912356/ PIP-47Bb SEQ ID NO: 4P. putida US20160186204A1 MNAPGAAPSEKEVAHAWLEGKARVKSTTAHGNIFAHDYNHPHQLTSTGRAMRTGADAINPFF SPAAGAATDSYANDANKNVSPGKAPVSIYEGLQTAIDIARRRSEYNPLDQPTDQRPKAKGDR EHFIAFTQQIAEIPFLSLLAAQVTQIQQKSHDANALIDSFVKGFIGLAAKDVEQIKKSLSSL VNAALSYSEQTERQSNFNQNILQTGIAGSVNFMLYASEFTIKATSKKGTITFQSSYTLSQAV YQLSVESWENVRDVFAKQQKTDTQQWLGDTTTPVKPGSSLRAICLVS (SEQ ID NO: 83) 14912356/ PIP-47Ba SEQ ID NO: 6P. fulva US20160186204A1 MHAPTVKELAHAWLTETTAKANSTIVRGNIFAHEYNHQLLTPTGLSMRSGADAINPFYSPAS GAATDSYAKDANNNVSPGSAPVSIYEGLQTSIDIARRRSGYNPLDQPTDQKPKAAGDREHFI AFTQQIANIPFLSLLAAQVTQIQQKSHDANALVDSFVKGFIGLKNQDVEQIKQSLSSLVNAA LSYSEQTERQSNFNQNILQTGNGGSVNFMLYASEFTIKASSHKGTITFQSSYTLSQAIYQLS VESWNNVKDTFSKQQKTDTEQWLDDTTTPVKEGSKLRAICLVG (SEQ ID NO: 84) 14912356/ PIP47Fa SEQ ID NO: 8P. chlororaphis US20160186204A1 MSTQNHKHITEKTLAWLNTTHESNKLSTQTNPNIFVLDRSRSSFSESLLTPGSRADIANPFF APAGSLATARYLQAANNNASSGSAPTSLQDGLQTCVNMARTRSGWNPNDPPTAANPHTTGDY EHFISFTKEISRIPFLTLESASSSLVMQQSHNADDLINSFANGFHGLETADIEETKRGLKEL VKAALSECEKTNRESFFNQHTLQQKDDTAIYLIYSSTFSIVATDQKGTINFQSSYLLTQSKY TLSNATWDRIKDLFYDQQKTDTNTWLNGMKTLPRAGSTARATCLEGQ (SEQ ID NO: 85) 15022109/ PIP-72Aa SEQ ID NO: 2P. chlororaphis US20160366891A1 MGITVTNNSSNPIEVAINHWGSDGDTSFFSVGNGKQETWDRSDSRGFVLSLKKNGAQHPYYV QASSKIEVDNNAVKDQGRLIEPLS (SEQ ID NO: 86)Monalysin from Monalysin MTIKEELGQPQSHSIELDEVSKEAASTRAAL P. entomophilaOpota et al. is also TSNLSGRFDQYPTKKGDFAIDGYLLDYSSPK derived from aQGCWVDGITVYGDIYIGKQNWGTYTRPVFAY PseudomonasLQYVETISIPQNVTTTLSYQLTKGHTRSFET SVNAKYSVGANIDIVNVGSEISTGFIRSESWSTTQSFTDTTEMKGPGTFVIYQVVLVYAHNA TSAGRQNANAFAYSKTQAVGSRVDLYYLSAITQRKRVIVPSSNAVTPLDWDTVQRNVLMENY NPGSNSGHFSFDWSAYNDPHRRY (SEQ ID NO: 87)¹All of the publications in Table 1 are incorporated herein byreference. ²SEQ ID NO from original source application/publicationdisplayed before sequence, SEQ ID NO according to current publicationdisplayed after sequence and underlined.

Insecticidal Proteins—Cry Proteins

Bacillus thuringiensis (Bt) are gram-positive spore-forming bacteriawith entomopathogenic properties. Bt produce insecticidal proteinsduring the sporulation phase as parasporal crystals. These crystals arepredominantly comprised of one or more proteins (Cry and Cyt toxins),also called δ-endotoxins. Cry proteins are parasporal inclusion(Crystal) proteins from Bacillus thuringiensis that exhibitexperimentally verifiable toxic effect to a target organism or havesignificant sequence similarity to a known Cry protein. Similarly, Cytproteins are parasporal inclusion proteins from Bacillus thuringiensisthat exhibit hemolytic (Cytolitic) activity or has obvious sequencesimilarity to a known Cyt protein. These toxins are highly specific totheir target insect, are innocuous to humans, vertebrates and plants,and are completely biodegradable. Bravo A, Gill S S, Soberón M., “Modeof action of Bacillus thuringiensis Cry and Cyt toxins and theirpotential for insect control,” Toxicon: Official Journal of theInternational Society on Toxinology. 2007; 49(4):423-435.

Bt Cry and Cyt toxins belong to a class of bacterial toxins known aspore-forming toxins (PFT) that are secreted as water-soluble proteinsundergoing conformational changes in order to insert into, or totranslocate across, cell membranes of their host. There are two maingroups of PFT: (i) the α-helical toxins, in which α-helix regions formthe trans-membrane pore, and (ii) the β-barrel toxins, that insert intothe membrane by forming a β-barrel composed of β-sheet hairpins fromeach monomer. See, Parker M W, Feil S C, “Pore-forming protein toxins:from structure to function,” Prog. Biophys. Mol. Biol. 2005 May;88(1):91-142. The first class of PFT includes toxins such as thecolicins, exotoxin A, diphtheria toxin and also the Cry three-domaintoxins. On the other hand, aerolysin, α-hemolysin, anthrax protectiveantigen, cholesterol-dependent toxins as the perfringolysin O and theCyt toxins belong to the β-barrel toxins. Id. In general, PFT-producingbacteria secrete their toxins and these toxins interact with specificreceptors located on the host cell surface. In most cases, PFT areactivated by host proteases after receptor binding inducing theformation of an oligomeric structure that is insertion competent.Finally, membrane insertion is triggered, in most cases, by a decreasein pH that induces a molten globule state of the protein. Id.

The development of transgenic crops that produce Bt Cry proteins hasallowed the substitution of chemical insecticides by environmentallyfriendly alternatives. In transgenic plants the Cry toxin is producedcontinuously, protecting the toxin from degradation and making itreachable to chewing and boring insects. Cry protein production inplants has been improved by engineering cry genes with a plant biasedcodon usage, by removal of putative splicing signal sequences anddeletion of the carboxy-terminal region of the protoxin. See, Schuler TH, et al., “Insect-resistant transgenic plants,” Trends Biotechnol.1998; 16:168-175. The use of insect resistant crops has diminishedconsiderably the use of chemical pesticides in areas where thesetransgenic crops are planted. See, Qaim M, Zilberman D, “Yield effectsof genetically modified crops in developing countries,” Science. 2003Feb. 7; 299(5608):900-902.

Known Cry proteins include: δ-endotoxins including but not limited to:the Cry1, Cry2, Cry3, Cry4, Cry5, Cry6, Cry7, Cry8, Cry9, Cry10, Cry11,Cry12, Cry13, Cry14, Cry15, Cry16, Cry17, Cry18, Cry19, Cry20, Cry21,Cry22, Cry23, Cry24, Cry25, Cry26, Cry27, Cry 28, Cry 29, Cry 30, Cry31,Cry32, Cry33, Cry34, Cry35, Cry36, Cry37, Cry38, Cry39, Cry40, Cry41,Cry42, Cry43, Cry44, Cry45, Cry 46, Cry47, Cry49, Cry 51, Cry52, Cry 53,Cry 54, Cry55, Cry56, Cry57, Cry58, Cry59. Cry60, Cry61, Cry62, Cry63,Cry64, Cry65, Cry66, Cry67, Cry68, Cry69, Cry70 and Cry71 classes ofδ-endotoxin genes and the B. thuringiensis cytolytic cyt1 and cyt2genes.

Members of these classes of B. thuringiensis insecticidal proteinsinclude, but are not limited to: Cry1Aa1 (Accession # AAA22353); Cry1Aa2(Accession # AAA22552); Cry1Aa3 (Accession # BAA00257); Cry1Aa4(Accession # CAA31886); Cry1Aa5 (Accession # BAA04468); Cry1Aa6(Accession # AAA86265); Cry1Aa7 (Accession # AAD46139); Cry1Aa8(Accession #126149); Cry1Aa9 (Accession # BAA77213); Cry1Aa10 (Accession# AAD55382); Cry1Aa11 (Accession # CAA70856); Cry1Aa12 (Accession #AAP80146); Cry1Aa13 (Accession # AAM44305); Cry1Aa14 (Accession #AAP40639); Cry1Aa15 (Accession # AAY66993); Cry1Aa16 (Accession #HQ439776); Cry1Aa17 (Accession # HQ439788); Cry1Aa18 (Accession #HQ439790); Cry1Aa19 (Accession # HQ685121); Cry1Aa20 (Accession #JF340156); Cry1Aa21 (Accession # JN651496); Cry1Aa22 (Accession #KC158223); Cry1Ab1 (Accession # AAA22330); Cry1Ab2 (Accession #AAA22613); Cry1Ab3 (Accession # AAA22561); Cry1Ab4 (Accession #BAA00071); Cry1Ab5 (Accession # CAA28405); Cry1Ab6 (Accession #AAA22420); Cry1Ab7 (Accession # CAA31620); Cry1Ab8 (Accession #AAA22551); Cry1Ab9 (Accession # CAA38701); Cry1Ab10 (Accession #A29125); Cry1Ab11 (Accession #112419); Cry1Ab12 (Accession # AAC64003);Cry1Ab13 (Accession # AAN76494); Cry1Ab14 (Accession # AAG16877);Cry1Ab15 (Accession # AA013302); Cry1Ab16 (Accession #AAK55546);Cry1Ab17 (Accession # AAT46415); Cry1Ab18 (Accession # AAQ88259);Cry1Ab19 (Accession # AAW31761); Cry1Ab20 (Accession # ABB72460);Cry1Ab21 (Accession # ABS18384); Cry1Ab22 (Accession # ABW87320);Cry1Ab23 (Accession # HQ439777); Cry1Ab24 (Accession # HQ439778);Cry1Ab25 (Accession # HQ685122); Cry1Ab26 (Accession # HQ847729);Cry1Ab27 (Accession # JN135249); Cry1Ab28 (Accession # JN135250);Cry1Ab29 (Accession # JN135251); Cry1Ab30 (Accession # JN135252);Cry1Ab31 (Accession # JN135253); Cry1Ab32 (Accession # JN135254);Cry1Ab33 (Accession # AAS93798); Cry1Ab34 (Accession # KC156668);Cry1Ab-like (Accession # AAK14336); Cry1Ab-like (Accession # AAK14337);Cry1Ab-like (Accession # AAK14338); Cry1Ab-like (Accession # ABG88858);Cry1Ac1 (Accession # AAA22331); Cry1Ac2 (Accession # AAA22338); Cry1Ac3(Accession # CAA38098); Cry1Ac4 (Accession # AAA73077); Cry1Ac5(Accession # AAA22339); Cry1Ac6 (Accession #AAA86266); Cry1Ac7(Accession # AAB46989); Cry1Ac8 (Accession # AAC44841); Cry1Ac9(Accession # AAB49768); Cry1Ac10 (Accession # CAA05505); Cry1Ac11(Accession # CAA10270); Cry1Ac12 (Accession #112418); Cry1Ac13(Accession # AAD38701); Cry1Ac14 (Accession # AAQ06607); Cry1Ac15(Accession # AAN07788); Cry1Ac16 (Accession # AAU87037); Cry1Ac17(Accession # AAX18704); Cry1Ac18 (Accession # AAY88347); Cry1Ac19(Accession # ABD37053); Cry1Ac20 (Accession # ABB89046); Cry1Ac21(Accession # AAY66992); Cry1Ac22 (Accession # ABZ01836); Cry1Ac23(Accession # CAQ30431); Cry1Ac24 (Accession # ABL01535); Cry1Ac25(Accession # FJ513324); Cry1Ac26 (Accession # FJ617446); Cry1Ac27(Accession # FJ617447); Cry1Ac28 (Accession # ACM90319); Cry1Ac29(Accession # DQ438941); Cry1Ac30 (Accession # GQ227507); Cry1Ac31(Accession # GU446674); Cry1Ac32 (Accession # HM061081); Cry1Ac33(Accession # GQ866913); Cry1Ac34 (Accession # HQ230364); Cry1Ac35(Accession # JF340157); Cry1Ac36 (Accession # JN387137); Cry1Ac37(Accession # JQ317685); Cry1Ad1 (Accession # AAA22340); Cry1Ad2(Accession # CAA01880); Cry1Ae1 (Accession # AAA22410); Cry1Af1(Accession # AAB82749); Cry1Ag1 (Accession # AAD46137); Cry1Ah1(Accession # AAQ14326); Cry1Ah2 (Accession # ABB76664); Cry1Ah3(Accession # HQ439779); Cry1Ai1 (Accession # AA039719); Cry1Ai2(Accession # HQ439780); Cry1A-like (Accession # AAK14339); Cry1Ba1(Accession # CAA29898); Cry1Ba2 (Accession # CAA65003); Cry1Ba3(Accession # AAK63251); Cry1Ba4 (Accession # AAK51084); Cry1Ba5(Accession # AB020894); Cry1Ba6 (Accession # ABL60921); Cry1Ba7(Accession # HQ439781); Cry1Bb1 (Accession # AAA22344); Cry1Bb2(Accession # HQ439782); Cry1Bc1 (Accession # CAA86568); Cry1Bd1(Accession # AAD10292); Cry1Bd2 (Accession # AAM93496); Cry1Be1(Accession # AAC32850); Cry1Be2 (Accession # AAQ52387); Cry1Be3(Accession # ACV96720); Cry1Be4 (Accession # HM070026); Cry1Bf1(Accession # CAC50778); Cry1Bf2 (Accession # AAQ52380); Cry1Bg1(Accession # AA039720); Cry1Bh1 (Accession # HQ589331); Cry1Bi1(Accession # KC156700); Cry1Ca1 (Accession # CAA30396); Cry1Ca2(Accession # CAA31951); Cry1Ca3 (Accession # AAA22343); Cry1Ca4(Accession # CAA01886); Cry1Ca5 (Accession # CAA65457); Cry1Ca6 [1](Accession # AAF37224); Cry1Ca7 (Accession # AAG50438); Cry1Ca8(Accession # AAM00264); Cry1Ca9 (Accession # AAL79362); Cry1Ca10(Accession # AAN16462); Cry1Ca11 (Accession # AAX53094); Cry1Ca12(Accession # HM070027); Cry1Ca13 (Accession # HQ412621); Cry1Ca14(Accession #JN651493); Cry1Cb1 (Accession # M97880); Cry1Cb2 (Accession# AAG35409); Cry1Cb3 (Accession # ACD50894); Cry1Cb-like (Accession #AAX63901); Cry1Da1 (Accession # CAA38099); Cry1Da2 (Accession #176415);Cry1Da3 (Accession # HQ439784); Cry1 Db1 (Accession # CAA80234); Cry1Db2 (Accession # AAK48937); Cry1 Dc1 (Accession # ABK35074); Cry1Ea1(Accession # CAA37933); Cry1Ea2 (Accession# CAA39609); Cry1Ea3(Accession # AAA22345); Cry1Ea4 (Accession # AAD04732); Cry1Ea5(Accession # A15535); Cry1Ea6 (Accession # AAL50330); Cry1Ea7 (Accession# AAW72936); Cry1Ea8 (Accession # ABX11258); Cry1Ea9 (Accession #HQ439785); Cry1Ea10 (Accession # ADR00398); Cry1Ea11 (Accession #JQ652456); Cry1Eb1 (Accession # AAA22346); Cry1Fa1 (Accession #AAA22348); Cry1Fa2 (Accession# AAA22347); Cry1Fa3 (Accession #HM070028); Cry1Fa4 (Accession #HM439638); Cry1Fb1 (Accession #CAA80235); Cry1Fb2 (Accession# BAA25298); Cry1Fb3 (Accession# AAF21767);Cry1Fb4 (Accession# AAC10641); Cry1Fb5 (Accession # AA013295); Cry1Fb6(Accession # ACD50892); Cry1Fb7 (Accession # ACD50893); Cry1Ga1(Accession # CAA80233); Cry1Ga2 (Accession # CAA70506); Cry1Gb1(Accession # AAD10291); Cry1Gb2 (Accession # AA013756); Cry1Gc1(Accession # AAQ52381); Cry1Ha1 (Accession# CAA80236); Cry1Hb1(Accession # AAA79694); Cry1Hb2 (Accession # HQ439786); Cry1H-like(Accession # AAF01213); Cry1Ia1 (Accession # CAA44633); Cry1Ia2(Accession # AAA22354); Cry1Ia3 (Accession # AAC36999); Cry1Ia4(Accession # AAB00958); Cry1Ia5 (Accession # CAA70124); Cry1Ia6(Accession # AAC26910); Cry1Ia7 (Accession # AAM73516); Cry1Ia8(Accession # AAK66742); Cry1Ia9 (Accession# AAQ08616); Cry1Ia10(Accession # AAP86782); Cry1Ia11 (Accession # CAC85964); Cry1Ia12(Accession # AAV53390); Cry1Ia13 (Accession # ABF83202); Cry1Ia14(Accession # ACG63871); Cry1Ia15 (Accession #FJ617445); Cry1Ia16(Accession # FJ617448); Cry1Ia17 (Accession # GU989199); Cry1Ia18(Accession # ADK23801); Cry1Ia19 (Accession # HQ439787); Cry1Ia20(Accession # JQ228426); Cry1Ia21 (Accession # JQ228424); Cry1Ia22(Accession #JQ228427); Cry1Ia23 (Accession # JQ228428); Cry1Ia24(Accession # JQ228429); Cry1Ia25 (Accession # JQ228430); Cry1Ia26(Accession # JQ228431); Cry1Ia27 (Accession # JQ228432); Cry1Ia28(Accession # JQ228433); Cry1Ia29 (Accession #JQ228434); Cry1Ia30(Accession# JQ317686); Cry1Ia31 (Accession # JX944038); Cry1Ia32(Accession # JX944039); Cry1Ia33 (Accession # JX944040); Cry1Ib1(Accession # AAA82114); Cry1Ib2 (Accession # ABW88019); Cry1Ib3(Accession # ACD75515); Cry1Ib4 (Accession # HM051227); Cry1Ib5(Accession # HM070028); Cry1Ib6 (Accession # ADK38579); Cry1Ib7(Accession # JN571740); Cry1Ib8 (Accession # JN675714); Cry1Ib9(Accession # JN675715); Cry1Ib10 (Accession # JN675716); Cry1Ib11(Accession # JQ228423); Cry1Ic1 (Accession # AAC62933); Cry1Ic2(Accession # AAE71691); Cry1Id1 (Accession # AAD44366); Cry1Id2(Accession # JQ228422); Cry1Ie1 (Accession # AAG43526); Cry1Ie2(Accession # HM439636); Cry1Ie3 (Accession # KC156647); Cry1Ie4(Accession # KC156681); Cry1If1 (Accession # AAQ52382); Cry1Ig1(Accession# KC156701); Cry1I-like (Accession # AAC31094); Cry1I-like(Accession # ABG88859); Cry1Ja1 (Accession # AAA22341); Cry1Ja2(Accession # HM070030); Cry1Ja3 (Accession # JQ228425); Cry1Jb1(Accession # AAA98959); Cry1Jc1 (Accession # AAC31092); Cry1Jc2(Accession # AAQ52372); Cry1Jd1 (Accession# CAC50779); Cry1Ka1(Accession # AAB00376); Cry1Ka2 (Accession # HQ439783); Cry1La1(Accession# AAS60191); Cry1La2 (Accession # HM070031); Cry1Ma1(Accession # FJ884067); Cry1Ma2 (Accession # KC156659); Cry1Na1(Accession # KC156648); Cry1Nb1 (Accession # KC156678); Cry1-like(Accession # AAC31091); Cry2Aa1 (Accession # AAA22335); Cry2Aa2(Accession # AAA83516); Cry2Aa3 (Accession # D86064); Cry2Aa4 (Accession# AAC04867); Cry2Aa5 (Accession # CAA10671); Cry2Aa6 (Accession #CAA10672); Cry2Aa7 (Accession # CAA10670); Cry2Aa8 (Accession #AA013734); Cry2Aa9 (Accession # AA013750); Cry2Aa10 (Accession #AAQ04263); Cry2Aa11 (Accession # AAQ52384); Cry2Aa12 (Accession #AB183671); Cry2Aa13 (Accession # ABL01536); Cry2Aa14 (Accession #ACF04939); Cry2Aa15 (Accession # JN426947); Cry2Ab1 (Accession #AAA22342); Cry2Ab2 (Accession # CAA39075); Cry2Ab3 (Accession #AAG36762); Cry2Ab4 (Accession # AA013296); Cry2Ab5 (Accession #AAQ04609); Cry2Ab6 (Accession # AAP59457); Cry2Ab7 (Accession #AAZ66347); Cry2Ab8 (Accession # ABC95996); Cry2Ab9 (Accession #ABC74968); Cry2Ab10 (Accession # EF157306); Cry2Ab11 (Accession #CAM84575); Cry2Ab12 (Accession # ABM21764); Cry2Ab13 (Accession #ACG76120); Cry2Ab14 (Accession # ACG76121); Cry2Ab15 (Accession #HM037126); Cry2Ab16 (Accession # GQ866914); Cry2Ab17 (Accession #HQ439789); Cry2Ab18 (Accession # JN135255); Cry2Ab19 (Accession #JN135256); Cry2Ab20 (Accession # JN135257); Cry2Ab21 (Accession #JN135258); Cry2Ab22 (Accession # JN135259); Cry2Ab23 (Accession #JN135260); Cry2Ab24 (Accession # JN135261); Cry2Ab25 (Accession #JN415485); Cry2Ab26 (Accession # JN426946); Cry2Ab27 (Accession #JN415764); Cry2Ab28 (Accession # JN651494); Cry2Ac1 (Accession #CAA40536); Cry2Ac2 (Accession # AAG35410); Cry2Ac3 (Accession #AAQ52385); Cry2Ac4 (Accession # ABC95997); Cry2Ac5 (Accession #ABC74969); Cry2Ac6 (Accession # ABC74793); Cry2Ac7 (Accession #CAL18690); Cry2Ac8 (Accession # CAM09325); Cry2Ac9 (Accession #CAM09326); Cry2Ac10 (Accession # ABN15104); Cry2Ac11 (Accession #CAM83895); Cry2Ac12 (Accession# CAM83896); Cry2Ad1 (Accession #AAF09583); Cry2Ad2 (Accession # ABC86927); Cry2Ad3 (Accession #CAK29504); Cry2Ad4 (Accession # CAM32331); Cry2Ad5 (Accession #CA078739); Cry2Ae1 (Accession # AAQ52362); Cry2Af1 (Accession #AB030519); Cry2Af2 (Accession # GQ866915); Cry2Ag1 (Accession #ACH91610); Cry2Ah1 (Accession # EU939453); Cry2Ah2 (Accession #ACL80665); Cry2Ah3 (Accession # GU073380); Cry2Ah4 (Accession #KC156702); Cry2Ai1 (Accession # FJ788388); Cry2Aj (Accession #); Cry2Ak1(Accession # KC156660); Cry2Ba1 (Accession# KC156658); Cry3Aa1(Accession# AAA22336); Cry3Aa2 (Accession # AAA22541); Cry3Aa3(Accession # CAA68482); Cry3Aa4 (Accession # AAA22542); Cry3Aa5(Accession # AAA50255); Cry3Aa6 (Accession # AAC43266); Cry3Aa7(Accession # CAB41411); Cry3Aa8 (Accession# AAS79487); Cry3Aa9(Accession # AAW05659); Cry3Aa10 (Accession #AAU29411); Cry3Aa11(Accession # AAW82872); Cry3Aa12 (Accession # ABY49136); Cry3Ba1(Accession # CAA34983); Cry3Ba2 (Accession # CAA00645); Cry3Ba3(Accession # JQ397327); Cry3Bb1 (Accession # AAA22334); Cry3Bb2(Accession # AAA74198); Cry3Bb3 (Accession #115475); Cry3Ca1 (Accession# CAA42469); Cry4Aa1 (Accession # CAA68485); Cry4Aa2 (Accession #BAA00179); Cry4Aa3 (Accession #CAD30148); Cry4Aa4 (Accession #AFB18317); Cry4A-like (Accession # AAY96321); Cry4Ba1 (Accession #CAA30312); Cry4Ba2 (Accession # CAA30114); Cry4Ba3 (Accession #AAA22337); Cry4Ba4 (Accession # BAA00178); Cry4Ba5 (Accession #CAD30095); Cry4Ba-like (Accession # ABC47686); Cry4Ca1 (Accession #EU646202); Cry4Cb1 (Accession # FJ403208); Cry4Cb2 (Accession #FJ597622); Cry4Cc1 (Accession # FJ403207); Cry5Aa1 (Accession #AAA67694); Cry5Ab1 (Accession # AAA67693); Cry5Ac1 (Accession #134543);Cry5Ad1 (Accession # ABQ82087); Cry5Ba1 (Accession # AAA68598); Cry5Ba2(Accession # ABW88931); Cry5Ba3 (Accession # AFJ04417); Cry5Ca1(Accession # HM461869); Cry5Ca2 (Accession # ZP_04123426); Cry5Da1(Accession # HM461870); Cry5Da2 (Accession # ZP_04123980); Cry5Ea1(Accession # HM485580); Cry5Ea2 (Accession # ZP_04124038); Cry6Aa1(Accession # AAA22357); Cry6Aa2 (Accession # AAM46849); Cry6Aa3(Accession # ABH03377); Cry6Ba1 (Accession # AAA22358); Cry7 Aa1(Accession # AAA22351); Cry7Ab1 (Accession # AAA21120); Cry7Ab2(Accession # AAA21121); Cry7Ab3 (Accession # ABX24522); Cry7Ab4(Accession # EU380678); Cry7Ab5 (Accession # ABX79555); Cry7Ab6(Accession# ACI44005); Cry7Ab7 (Accession# ADB89216); Cry7Ab8 (Accession# GU145299); Cry7Ab9 (Accession # ADD92572); Cry7Ba1 (Accession #ABB70817); Cry7Bb1 (Accession # KC156653); Cry7Ca1 (Accession #ABR67863); Cry7Cb1 (Accession # KC156698); Cry7Da1 (Accession #ACQ99547); Cry7Da2 (Accession # HM572236); Cry7Da3 (Accession#KC156679); Cry7Ea1 (Accession #HM035086); Cry7Ea2 (Accession #HM132124); Cry7Ea3 (Accession # EEM19403); Cry7Fa1 (Accession #HM035088); Cry7Fa2 (Accession # EEM19090); Cry7Fb1 (Accession #HM572235); Cry7Fb2 (Accession # KC156682); Cry7Ga1 (Accession #HM572237); Cry7Ga2 (Accession # KC156669); Cry7Gb1 (Accession #KC156650); Cry7Gc1 (Accession # KC156654); Cry7Gd1 (Accession #KC156697); Cry7Ha1 (Accession # KC156651); Cry7Ia1 (Accession #KC156665); Cry7Ja1 (Accession # KC156671); Cry7Ka1 (Accession #KC156680); Cry7Kb1 (Accession # BAM99306); Cry7La1 (Accession #BAM99307); Cry8Aa1 (Accession # AAA21117); Cry8Ab1 (Accession #EU044830); Cry8Ac1 (Accession # KC156662); Cry8Ad1 (Accession #KC156684); Cry8Ba1 (Accession # AAA21118); Cry8Bb1 (Accession #CAD57542); Cry8Bc1 (Accession # CAD57543); Cry8Ca1 (Accession #AAA21119); Cry8Ca2 (Accession # AAR98783); Cry8Ca3 (Accession #EU625349); Cry8Ca4 (Accession # ADB54826); Cry8Da1 (Accession #BAC07226); Cry8Da2 (Accession # BD133574); Cry8Da3 (Accession #BD133575); Cry8Db1 (Accession # BAF93483); Cry8Ea1 (Accession #AAQ73470); Cry8Ea2 (Accession # EU047597); Cry8Ea3 (Accession #KC855216); Cry8Fa1 (Accession # AAT48690); Cry8Fa2 (Accession #HQ174208); Cry8Fa3 (Accession # AFH78109); Cry8Ga1 (Accession #AAT46073); Cry8Ga2 (Accession # ABC42043); Cry8Ga3 (Accession #FJ198072); Cry8Ha1 (Accession # AAW81032); Cry8Ia1 (Accession #EU381044); Cry8Ia2 (Accession # GU073381); Cry8Ia3 (Accession #HM044664); Cry8Ia4 (Accession # KC156674); Cry8Ib1 (Accession #GU325772); Cry8Ib2 (Accession # KC156677); Cry8Ja1 (Accession #EU625348); Cry8Ka1 (Accession # FJ422558); Cry8Ka2 (Accession #ACN87262); Cry8Kb1 (Accession # HM123758); Cry8Kb2 (Accession #KC156675); Cry8La1 (Accession # GU325771); Cry8Ma1 (Accession #HM044665); Cry8Ma2 (Accession # EEM86551); Cry8Ma3 (Accession #HM210574); Cry8Na1 (Accession # HM640939); Cry8Pa1 (Accession #HQ388415); Cry8Qa1 (Accession # HQ441166); Cry8Qa2 (Accession #KC152468); Cry8Ra1 (Accession # AFP87548); Cry8Sa1 (Accession #JQ740599); Cry8Ta1 (Accession # KC156673); Cry8-like (Accession #FJ770571); Cry8-like (Accession # ABS53003); Cry9Aa1 (Accession #CAA41122); Cry9Aa2 (Accession # CAA41425); Cry9Aa3 (Accession #GQ249293); Cry9Aa4 (Accession # GQ249294); Cry9Aa5 (Accession #JX174110); Cry9Aa like (Accession # AAQ52376); Cry9Ba1 (Accession #CAA52927); Cry9Ba2 (Accession # GU299522); Cry9Bb1 (Accession #AAV28716); Cry9Ca1 (Accession # CAA85764); Cry9Ca2 (Accession #AAQ52375); Cry9Da1 (Accession # BAA19948); Cry9Da2 (Accession #AAB97923); Cry9Da3 (Accession # GQ249293); Cry9Da4 (Accession #GQ249297); Cry9Db1 (Accession # AAX78439); Cry9Dc1 (Accession#KC156683); Cry9Ea1 (Accession # BAA34908); Cry9Ea2 (Accession #AA012908); Cry9Ea3 (Accession# ABM21765); Cry9Ea4 (Accession #ACE88267); Cry9Ea5 (Accession # ACF04743); Cry9Ea6 (Accession#ACG63872); Cry9Ea7 (Accession # FJ380927); Cry9Ea8 (Accession #GQ249292); Cry9Ea9 (Accession # JN651495); Cry9Eb1 (Accession #CAC50780); Cry9Eb2 (Accession # GQ249298); Cry9Eb3 (Accession #KC156646); Cry9Ec1 (Accession # AAC63366); Cry9Ed1 (Accession #AAX78440); Cry9Ee1 (Accession # GQ249296); Cry9Ee2 (Accession #KC156664); Cry9Fa1 (Accession # KC156692); Cry9Ga1 (Accession #KC156699); Cry9-like (Accession # AAC63366); Cry10Aa1 (Accession#AAA22614); Cry10Aa2 (Accession # E00614); Cry10Aa3 (Accession #CAD30098); Cry10Aa4 (Accession # AFB18318); Cry10A-like (Accession #DQ167578); Cry11Aa1 (Accession # AAA22352); Cry11Aa2 (Accession #AAA22611); Cry11Aa3 (Accession # CAD30081); Cry11Aa4 (Accession#AFB18319); Cry11Aa-like (Accession # DQ166531); Cry1 1Ba1 (Accession #CAA60504); Cry1 1Bb1 (Accession # AAC97162); Cry1 1Bb2 (Accession #HM068615); Cry12Aa1 (Accession # AAA22355); Cry13Aa1 (Accession #AAA22356); Cry14Aa1 (Accession # AAA21516); Cry14Ab1 (Accession #KC156652); Cry15Aa1 (Accession # AAA22333); Cry16Aa1 (Accession #CAA63860); Cry17Aa1 (Accession # CAA67841); Cry18Aa1 (Accession #CAA67506); Cry18Ba1 (Accession # AAF89667); Cry18Ca1 (Accession #AAF89668); Cry19Aa1 (Accession # CAA68875); Cry19Ba1 (Accession #BAA32397); Cry19Ca1 (Accession # AFM37572); Cry20Aa1 (Accession #AAB93476); Cry20Ba1 (Accession # ACS93601); Cry20Ba2 (Accession #KC156694); Cry20-like (Accession # GQ144333); Cry21Aa1 (Accession#132932); Cry21Aa2 (Accession #166477); Cry21Ba1 (Accession # BAC06484);Cry21Ca1 (Accession # JF521577); Cry21Ca2 (Accession # KC156687);Cry21Da1 (Accession # JF521578); Cry22Aa1 (Accession #134547); Cry22Aa2(Accession # CAD43579); Cry22Aa3 (Accession # ACD93211); Cry22Ab1(Accession # AAK50456); Cry22Ab2 (Accession # CAD43577); Cry22Ba1(Accession # CAD43578); Cry22Bb1 (Accession # KC156672); Cry23Aa1(Accession # AAF76375); Cry24Aa1 (Accession # AAC61891); Cry24Ba1(Accession # BAD32657); Cry24Ca1 (Accession # CAJ43600); Cry25Aa1(Accession # AAC61892); Cry26Aa1 (Accession # AAD25075); Cry27Aa1(Accession # BAA82796); Cry28Aa1 (Accession # AAD24189); Cry28Aa2(Accession # AAG00235); Cry29Aa1 (Accession # CAC80985); Cry30Aa1(Accession # CAC80986); Cry30Ba1 (Accession # BAD00052); Cry30Ca1(Accession # BAD67157); Cry30Ca2 (Accession # ACU24781); Cry30Da1(Accession # EF095955); Cry30Db1 (Accession # BAE80088); Cry30Ea1(Accession # ACC95445); Cry30Ea2 (Accession # FJ499389); Cry30Fa1(Accession # ACI22625); Cry30Ga1 (Accession # ACG60020); Cry30Ga2(Accession #HQ638217); Cry31Aa1 (Accession # BAB11757); Cry31Aa2(Accession # AAL87458); Cry31Aa3 (Accession # BAE79808); Cry31Aa4(Accession# BAF32571); Cry31Aa5 (Accession # BAF32572); Cry31Aa6(Accession # BA144026); Cry31Ab1 (Accession #BAE79809); Cry31Ab2(Accession # BAF32570); Cry31Ac1 (Accession # BAF34368); Cry31Ac2(Accession # AB731600); Cry31Ad1 (Accession # BA144022); Cry32Aa1(Accession # AAG36711); Cry32Aa2 (Accession # GU063849); Cry32Ab1(Accession #GU063850); Cry32Ba1 (Accession # BAB78601); Cry32Ca1(Accession # BAB78602); Cry32Cb1 (Accession # KC156708); Cry32 Da1(Accession # BAB78603); Cry32Ea1 (Accession # GU324274); Cry32Ea2(Accession # KC156686); Cry32Eb1 (Accession # KC156663); Cry32Fa1(Accession # KC156656); Cry32Ga1 (Accession # KC156657); Cry32Ha1(Accession # KC156661); Cry32Hb1 (Accession# KC156666); Cry32Ia1(Accession # KC156667); Cry32Ja1 (Accession # KC156685); Cry32Ka1(Accession # KC156688); Cry32La1 (Accession # KC156689); Cry32Ma1(Accession # KC156690); Cry32 Mb1 (Accession # KC156704); Cry32Na1(Accession # KC156691); Cry32Oa1 (Accession # KC156703); Cry32 Pa1(Accession# KC156705); Cry32Qa1 (Accession #KC156706); Cry32Ra1(Accession # KC156707); Cry32Sa1 (Accession # KC156709); Cry32Ta1(Accession # KC156710); Cry32Ua1 (Accession # KC156655); Cry33Aa1(Accession #AAL26871); Cry34Aa1 (Accession # AAG50341); Cry34Aa2(Accession #AAK64560); Cry34Aa3 (Accession # AAT29032); Cry34Aa4(Accession # AAT29030); Cry34Ab1 (Accession # AAG41671); Cry34Ac1(Accession # AAG50118); Cry34Ac2 (Accession # AAK64562); Cry34Ac3(Accession # AAT29029); Cry34Ba1 (Accession # AAK64565); Cry34Ba2(Accession # AAT29033); Cry34Ba3 (Accession # AAT29031); Cry35Aa1(Accession # AAG50342); Cry35Aa2 (Accession # AAK64561); Cry35Aa3(Accession # AAT29028); Cry35Aa4 (Accession # AAT29025); Cry35Ab1(Accession # AAG41672); Cry35Ab2 (Accession # AAK64563); Cry35Ab3(Accession # AY536891); Cry35Ac1 (Accession # AAG50117); Cry35Ba1(Accession # AAK64566); Cry35Ba2 (Accession # AAT29027); Cry35Ba3(Accession # AAT29026); Cry36Aa1 (Accession # AAK64558); Cry37Aa1(Accession # AAF76376); Cry38Aa1 (Accession # AAK64559); Cry39Aa1(Accession # BAB72016); Cry40Aa1 (Accession # BAB72018); Cry40Ba1(Accession # BAC77648); Cry40Ca1 (Accession # EU381045); Cry40Da1(Accession # ACF15199); Cry41Aa1 (Accession #BAD35157); Cry41Ab1(Accession #BAD35163); Cry41Ba1 (Accession # HM461871); Cry41Ba2(Accession # ZP_04099652); Cry42Aa1 (Accession # BAD35166); Cry43Aa1(Accession # BAD15301); Cry43Aa2 (Accession # BAD95474); Cry43Ba1(Accession # BAD15303); Cry43Ca1 (Accession # KC156676); Cry43Cb1(Accession # KC156695); Cry43Cc1 (Accession # KC156696); Cry43-like(Accession # BAD15305); Cry44Aa (Accession # BAD08532); Cry45Aa(Accession # BAD22577); Cry46Aa (Accession # BAC79010); Cry46Aa2(Accession # BAG68906); Cry46Ab (Accession # BAD35170); Cry47 Aa(Accession # AAY24695); Cry48Aa (Accession # CAJ18351); Cry48Aa2(Accession # CAJ86545); Cry48Aa3 (Accession # CAJ86546); Cry48Ab(Accession # CAJ86548); Cry48Ab2 (Accession # CAJ86549); Cry49Aa(Accession # CAH56541); Cry49Aa2 (Accession # CAJ86541); Cry49Aa3(Accession # CAJ86543); Cry49Aa4 (Accession # CAJ86544); Cry49Ab1(Accession # CAJ86542); Cry50Aa1 (Accession # BAE86999); Cry50Ba1(Accession # GU446675); Cry50Ba2 (Accession # GU446676); Cry51Aa1(Accession # AB114444); Cry51Aa2 (Accession # GU570697); Cry52Aa1(Accession # EF613489); Cry52Ba1 (Accession # FJ361760); Cry53Aa1(Accession # EF633476); Cry53Ab1 (Accession # FJ361759); Cry54Aa1(Accession # ACA52194); Cry54Aa2 (Accession# GQ140349); Cry54Ba1(Accession # GU446677); Cry55Aa1 (Accession # ABW88932); Cry54Ab1(Accession # JQ916908); Cry55Aa2 (Accession # AAE33526); Cry56Aa1(Accession # ACU57499); Cry56Aa2 (Accession # GQ483512); Cry56Aa3(Accession # JX025567); Cry57Aa1 (Accession # ANC87261); Cry58Aa1(Accession # ANC87260); Cry59Ba1 (Accession # JN790647); Cry59Aa1(Accession # ACR43758); Cry60Aa1 (Accession # ACU24782); Cry60Aa2(Accession # EA057254); Cry60Aa3 (Accession # EEM99278); Cry60Ba1(Accession # GU810818); Cry60Ba2 (Accession # EA057253); Cry60Ba3(Accession # EEM99279); Cry61Aa1 (Accession # HM035087); Cry61Aa2(Accession # HM132125); Cry61Aa3 (Accession # EEM19308); Cry62Aa1(Accession # HM054509); Cry63Aa1 (Accession # BA144028); Cry64Aa1(Accession # BAJ05397); Cry65Aa1 (Accession # HM461868); Cry65Aa2(Accession # ZP_04123838); Cry66Aa1 (Accession # HM485581); Cry66Aa2(Accession # ZP_04099945); Cry67Aa1 (Accession #HM485582); Cry67Aa2(Accession# ZP_04148882); Cry68Aa1 (Accession# HQ113114); Cry69Aa1(Accession # HQ401006); Cry69Aa2 (Accession # JQ821388); Cry69Ab1(Accession # JN209957); Cry70Aa1 (Accession # JN646781); Cry70Ba1(Accession # AD051070); Cry70Bb1 (Accession # EEL67276); Cry71Aa1(Accession # JX025568); Cry72Aa1 (Accession # JX025569); Cyt1Aa (GenBankAccession Number X03182); Cyt1Ab (GenBank Accession Number X98793);Cyt1B (GenBank Accession Number U37196); Cyt2A (GenBank Accession NumberZ14147); and Cyt2B (GenBank Accession Number U52043).

Examples of δ-endotoxins also include but are not limited to Cry1Aproteins of U.S. Pat. Nos. 5,880,275, 7,858,849 8,530,411, 8,575,433,and 8,686,233; a DIG-3 or DIG-11 toxin (N-terminal deletion of α-helix 1and/or α-helix 2 variants of cry proteins such as Cry1A, Cry3A) of U.S.Pat. Nos. 8,304,604, 8,304,605 and 8,476,226; Cry1B of U.S. patentapplication Ser. No. 10/525,318; Cry1C of U.S. Pat. No. 6,033,874; Cry1Fof U.S. Pat. Nos. 5,188,960 and 6,218,188; Cry1A/F chimeras of U.S. Pat.Nos. 7,070,982; 6,962,705 and 6,713,063); a Cry2 protein such as Cry2Abprotein of U.S. Pat. No. 7,064,249); a Cry3A protein including but notlimited to an engineered hybrid insecticidal protein (eHIP) created byfusing unique combinations of variable regions and conserved blocks ofat least two different Cry proteins (US Patent Application PublicationNumber 2010/0017914); a Cry4 protein; a Cry5 protein; a Cry6 protein;Cry8 proteins of U.S. Pat. Nos. 7,329,736, 7,449,552, 7,803,943,7,476,781, 7,105,332, 7,378,499 and 7,462,760; a Cry9 protein such assuch as members of the Cry9A, Cry9B, Cry9C, Cry9D, Cry9E and Cry9Ffamilies, including but not limited to the Cry9D protein of U.S. Pat.No. 8,802,933 and the Cry9B protein of U.S. Pat. No. 8,802,934; a Cry15protein of Naimov, et al., (2008), “Applied and EnvironmentalMicrobiology,” 74:7145-7151; a Cry22, a Cry34Ab1 protein of U.S. Pat.Nos. 6,127,180, 6,624,145 and 6,340,593; a CryET33 and CryET34 proteinof U.S. Pat. Nos. 6,248,535, 6,326,351, 6,399,330, 6,949,626, 7,385,107and 7,504,229; a CryET33 and CryET34 homologs of US Patent PublicationNumber 2006/0191034, 2012/0278954, and PCT Publication Number WO2012/139004; a Cry35Ab1 protein of U.S. Pat. Nos. 6,083,499, 6,548,291and 6,340,593; a Cry46 protein, a Cry51 protein, a Cry binary toxin; aTIC901 or related toxin; TIC807 of US Patent Application PublicationNumber 2008/0295207; ET29, ET37, TIC809, TIC810, TIC812, TIC127, TIC128of PCT US 2006/033867; TIC853 toxins of U.S. Pat. No. 8,513,494,AXMI-027, AXMI-036, and AXMI-038 of U.S. Pat. No. 8,236,757; AXMI-031,AXMI-039, AXMI-040, AXMI-049 of U.S. Pat. No. 7,923,602; AXMI-018,AXMI-020 and AXMI-021 of WO 2006/083891; AXMI-010 of WO 2005/038032;AXMI-003 of WO 2005/021585; AXMI-008 of US Patent ApplicationPublication Number 2004/0250311; AXMI-006 of US Patent ApplicationPublication Number 2004/0216186; AXMI-007 of US Patent ApplicationPublication Number 2004/0210965; AXMI-009 of US Patent ApplicationNumber 2004/0210964; AXMI-014 of US Patent Application PublicationNumber 2004/0197917; AXMI-004 of US Patent Application PublicationNumber 2004/0197916; AXMI-028 and AXMI-029 of WO 2006/119457; AXMI-007,AXMI-008, AXMI-008orf2, AXMI-009, AXMI-014 and AXMI-004 of WO2004/074462; AXMI-150 of U.S. Pat. No. 8,084,416; AXMI-205 of US PatentApplication Publication Number 2011/0023184; AXMI-011, AXMI-012,AXMI-013, AXMI-015, AXMI-019, AXMI-044, AXMI-037, AXMI-043, AXMI-033,AXMI-034, AXMI-022, AXMI-023, AXMI-041, AXMI-063 and AXMI-064 of USPatent Application Publication Number 2011/0263488; AXMI-R1 and relatedproteins of US Patent Application Publication Number 2010/0197592;AXMI221z, AXMI222z, AXMI223z, AXMI224z and AXMI225z of WO 2011/103248;AXMI218, AXMI219, AXMI220, AXMI226, AXMI227, AXMI228, AXMI229, AXMI230and AXMI231 of WO 2011/103247 and U.S. Pat. No. 8,759,619; AXMI-115,AXMI-113, AXMI-005, AXMI-163 and AXMI-184 of U.S. Pat. No. 8,334,431;AXMI-001, AXMI-002, AXMI-030, AXMI-035 and AXMI-045 of US PatentApplication Publication Number 2010/0298211; AXMI-066 and AXMI-076 of USPatent Application Publication Number 2009/0144852; AXMI128, AXMI130,AXMI131, AXMI133, AXMI140, AXMI141, AXMI142, AXMI143, AXMI144, AXMI146,AXMI148, AXMI149, AXMI152, AXMI153, AXMI154, AXMI155, AXMI156, AXMI157,AXMI158, AXMI162, AXMI165, AXMI166, AXMI167, AXMI168, AXMI169, AXMI170,AXMI171, AXMI172, AXMI173, AXMI174, AXMI175, AXMI176, AXMI177, AXMI178,AXMI179, AXMI180, AXMI181, AXMI182, AXMI185, AXMI186, AXMI187, AXMI188,AXMI189 of U.S. Pat. No. 8,318,900; AXMI079, AXMI080, AXMI081, AXMI082,AXMI091, AXMI092, AXMI096, AXMI097, AXMI098, AXMI099, AXMI100, AXMI101,AXMI102, AXMI103, AXMI104, AXMI107, AXMI108, AXMI109, AXMI110, AXMI111,AXMI112, AXMI114, AXMI116, AXMI117, AXMI118, AXMI119, AXMI120, AXMI121,AXMI122, AXMI123, AXMI124, AXMI1257, AXMI1268, AXMI127, AXMI129,AXMI164, AXMI151, AXMI161, AXMI183, AXMI132, AXMI138, AXMI137 of USPatent Application Publication Number 2010/0005543, AXMI270 of US PatentApplication Publication US20140223598, AXMI279 of US Patent ApplicationPublication US20140223599, cry proteins such as Cry1A and Cry3A havingmodified proteolytic sites of U.S. Pat. No. 8,319,019; a Cry1Ac, Cry2Aaand Cry1Ca toxin protein from Bacillus thuringiensis strain VBTS 2528 ofUS Patent Application Publication Number 2011/0064710.

Other Cry proteins are well known to one skilled in the art. See, N.Crickmore, et al., “Revision of the Nomenclature for the Bacillusthuringiensis Pesticidal Crystal Proteins,” Microbiology and MolecularBiology Reviews,” (1998) Vol 62: 807-813; see also, N. Crickmore, etal., “Bacillus thuringiensis toxin nomenclature” (2016), atbtnomenclature.info, which can be accessed on the worldwide web usingthe “www” prefix

The use of Cry proteins as transgenic plant traits is well known to oneskilled in the art and Cry-transgenic plants including but not limitedto plants expressing Cry1Ac, Cry1 Ac+Cry2Ab, Cry1Ab, Cry1A.105, Cry1F,Cry1Fa2, Cry1F+Cry1Ac, Cry2Ab, Cry3A, mCry3A, Cry3Bb1, Cry34Ab1,Cry35Ab1, Vip3A, mCry3A, Cry9c and CBI-Bt have received regulatoryapproval. See, Sanahuja et al., “Bacillus thuringiensis: a century ofresearch, development and commercial applications,” (2011) PlantBiotech. Journal, April 9(3):283-300 and the CERA (2010) GM CropDatabase Center for Environmental Risk Assessment (CERA), ILSI ResearchFoundation, Washington D.C. atcera-gmc.org/index.php?action=gm_crop_database, which can be accessed onthe worldwide web using the “www” prefix. More than one pesticidalproteins well known to one skilled in the art can also be expressed inplants such as Vip3Ab & Cry1Fa (US2012/0317682); Cry1BE & Cry1F(US2012/0311746); Cry1CA & Cry1AB (US2012/0311745); Cry1F & CryCa(US2012/0317681); Cry1Da& Cry1Be (U52012/0331590); Cry1DA & Cry1Fa(US2012/0331589); Cry1Ab & Cry1Be (U52012/0324606); Cry1Fa & Cry2Aa andCry1I & Cry1E (US2012/0324605); Cry34Ab/35Ab and Cry6Aa (US20130167269);Cry34Ab/VCry35Ab & Cry3Aa (US20130167268); Cry1Ab & Cry1F(U520140182018); and Cry3A and Cry1Ab or Vip3Aa (US20130116170).Pesticidal proteins also include insecticidal lipases including lipidacyl hydrolases of U.S. Pat. No. 7,491,869, and cholesterol oxidasessuch as from Streptomyces (Purcell et al. (1993) Biochem. Biophys. Res.Commun. 15:1406-1413).

Insecticidal Proteins—Vips

Pesticidal proteins also include Vip (vegetative insecticidal proteins)toxins.

As described in the art, “Entomopathogenic bacteria produce insecticidalproteins that accumulate in inclusion bodies or parasporal crystals(such as the aforementioned Cry and Cyt proteins), as well asinsecticidal proteins that are secreted into the culture medium. Amongthe latter are the Vip proteins, which are divided into four familiesaccording to their amino acid identity. The Vip1 and Vip2 proteins actas binary toxins and are toxic to some members of the Coleoptera andHemiptera. The Vip1 component is thought to bind to receptors in themembrane of the insect midgut, and the Vip2 component enters the cell,where it displays its ADP-ribosyltransferase activity against actin,preventing microfilament formation. Vip3 has no sequence similarity toVip1 or Vip2 and is toxic to a wide variety of members of theLepidoptera. Its mode of action has been shown to resemble that of theCry proteins in terms of proteolytic activation, binding to the midgutepithelial membrane, and pore formation, although Vip3A proteins do notshare binding sites with Cry proteins. The latter property makes themgood candidates to be combined with Cry proteins in transgenic plants(Bacillus thuringiensis-treated crops [Bt crops]) to prevent or delayinsect resistance and to broaden the insecticidal spectrum. There arecommercially grown varieties of Bt cotton and Bt maize that express theVip3Aa protein in combination with Cry proteins. For the most recentlyreported Vip4 family, no target insects have been found yet.” See,Chakroun et al., “Bacterial Vegetative Insecticidal Proteins (Vip) fromEntomopathogenic Bacteria,” Microbiol. Mol. Biol. Rev. 2016 Mar. 2;80(2):329-350.

VIPs can be found in U.S. Pat. Nos. 5,877,012, 6,107,279 6,137,033,7,244,820, 7,615,686, and 8,237,020 and the like. Other VIP proteins arewell known to one skilled in the art (see,lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/vip.html, which can beaccessed on the worldwide web using the “www” prefix).

Insecticidal Proteins—Toxin Complex (TC) Family Proteins

Pesticidal proteins also include toxin complex (TC) proteins, obtainablefrom organisms such as Xenorhabdus, Photorhabdus and Paenibacillus (see,U.S. Pat. Nos. 7,491,698 and 8,084,418). Some TC proteins have “standalone” insecticidal activity and other TC proteins enhance the activityof the stand-alone toxins produced by the same given organism. Thetoxicity of a “stand-alone” TC proteins (from Photorhabdus, Xenorhabdusor Paenibacillus, for example) can be enhanced by one or more TC protein“potentiators” derived from a source organism of a different genus.There are three main types of TC proteins. As referred to herein, ClassA proteins (“Protein A”) are stand-alone toxins. Class B proteins(“Protein B”) and Class C proteins (“Protein C”) enhance the toxicity ofClass A proteins. Examples of Class A proteins are TcbA, TcdA, XptA1 andXptA2. Examples of Class B proteins are TcaC, TcdB, XptBlXb and XptClWi.Examples of Class C proteins are TccC, XptClXb and XptBl Wi. Pesticidalproteins also include spider, snake and scorpion venom proteins.Examples of spider venom peptides include, but are not limited tolycotoxin-1 peptides and mutants thereof (U.S. Pat. No. 8,334,366).

Insecticidal Proteins—Combinations

In some embodiments, the disclosure contemplates utilizing a combinationof one or more insecticidal proteins. For example, it is known that Cryproteins have limited utility against all common agricultural pests, asthe proteins only target specific receptors found in susceptible insectspecies. Consequently, by expressing a Cry along with a novelinsecticidal protein as taught herein, it is contemplated that a plantspecies would have expanded protection against a broader class ofinsects.

The disclosure therefore contemplates engineered plant species thatproduce a novel insecticidal protein as taught herein, in combinationwith said plant species also expressing one or more other insecticidalproteins, e.g. Monalysin, PIP, Cry, Cyt, VIP, TC, and any combinationthereof.

Nucleic Acid Molecules Encoding Discovered Insecticidal Proteins

One aspect of the disclosure pertains to isolated or recombinant nucleicacid molecules comprising nucleic acid sequences encoding insecticidalpolypeptides, proteins, or biologically active portions thereof, as wellas nucleic acid molecules sufficient for use as hybridization probes toidentify nucleic acid molecules encoding proteins with regions ofsequence homology.

As used herein, the term “nucleic acid molecule” refers to DNA molecules(e.g., recombinant DNA, cDNA, genomic DNA, plastid DNA, mitochondrialDNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNAgenerated using nucleotide analogs. The nucleic acid molecule can besingle-stranded or double-stranded.

An “isolated” nucleic acid molecule (or DNA) is used herein to refer toa nucleic acid sequence (or DNA) that is no longer in its naturalenvironment, for example in vitro. A “recombinant” nucleic acid molecule(or DNA) is used herein to refer to a nucleic acid sequence (or DNA)that is in a recombinant bacterial or plant host cell. In someembodiments, an “isolated” or “recombinant” nucleic acid is free ofsequences that naturally flank the nucleic acid (i.e., sequences locatedat the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of theorganism from which the nucleic acid is derived. For purposes of thedisclosure, “isolated” or “recombinant” when used to refer to nucleicacid molecules excludes isolated chromosomes. For example, in variousembodiments, the recombinant nucleic acid molecule encoding aninsecticidal protein of the disclosure can contain less than about 5 kb,4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleic acid sequences thatnaturally flank the nucleic acid molecule in genomic DNA of the cellfrom which the nucleic acid is derived.

In some embodiments, an isolated nucleic acid molecule encoding aninsecticidal protein has one or more changes in the nucleic acidsequence compared to the native or genomic nucleic acid sequence. Insome embodiments, the change in the native or genomic nucleic acidsequence includes, but is not limited to: changes in the nucleic acidsequence due to the degeneracy of the genetic code; changes in thenucleic acid sequence due to the amino acid substitution, insertion,deletion, and/or addition compared to the native or genomic sequence;removal of one or more intron; deletion of one or more upstream ordownstream regulatory regions; and deletion of the 5′ and/or 3′untranslated region associated with the genomic nucleic acid sequence.In some embodiments, the nucleic acid molecule encoding an insecticidalprotein is a non-genomic sequence.

A variety of polynucleotides that encode an insecticidal protein of thedisclosure are contemplated. Such polynucleotides are useful forproduction of the insecticidal proteins in host cells when operablylinked to suitable promoter, transcription termination and/orpolyadenylation sequences. Such polynucleotides are also useful asprobes for isolating homologous or substantially homologouspolynucleotides that encode further insecticidal proteins.

Polynucleotides that encode an insecticidal protein of the disclosurecan be synthesized de novo from a sequence disclosed herein. Thesequence of the polynucleotide gene can be deduced from a disclosedprotein sequence through use of the genetic code. Computer programs suchas “BackTranslate” (GCG™ Package, Acclerys, Inc. San Diego, Calif.) canbe used to convert a peptide sequence to the corresponding nucleotidesequence encoding the peptide.

Furthermore, synthetic polynucleotide sequences of the disclosure can bedesigned so that they will be expressed in plants. U.S. Pat. No.5,500,365 describes a method for synthesizing plant genes to improve theexpression level of the protein encoded by the synthesized gene. Thismethod relates to the modification of the structural gene sequences ofthe exogenous transgene, to cause them to be more efficientlytranscribed, processed, translated and expressed by the plant. Featuresof genes that are expressed well in plants include elimination ofsequences that can cause undesired intron splicing or polyadenylation inthe coding region of a gene transcript while retaining substantially theamino acid sequence of the toxic portion of the insecticidal protein. Asimilar method for obtaining enhanced expression of transgenes inmonocotyledonous plants is disclosed in U.S. Pat. No. 5,689,052.“Complement” is used herein to refer to a nucleic acid sequence that issufficiently complementary to a given nucleic acid sequence such that itcan hybridize to the given nucleic acid sequence to thereby form astable duplex. “Polynucleotide sequence variants” is used herein torefer to a nucleic acid sequence that except for the degeneracy of thegenetic code encodes the same polypeptide.

In some embodiments, a nucleic acid molecule encoding an insecticidalprotein of the disclosure is a non-genomic nucleic acid sequence. Asused herein a “non-genomic nucleic acid sequence” or “non-genomicnucleic acid molecule” refers to a nucleic acid molecule that has one ormore changes in the nucleic acid sequence compared to a native orgenomic nucleic acid sequence. In some embodiments, the change to anative or genomic nucleic acid molecule includes but is not limited to:changes in the nucleic acid sequence due to the degeneracy of thegenetic code; codon optimization of the nucleic acid sequence forexpression in plants; changes in the nucleic acid sequence to introduceat least one amino acid substitution, insertion, deletion and/oraddition compared to the native or genomic sequence; removal of one ormore intron associated with the genomic nucleic acid sequence; insertionof one or more heterologous intrans; deletion of one or more upstream ordownstream regulatory regions associated with the genomic nucleic acidsequence; insertion of one or more heterologous upstream or downstreamregulatory regions; deletion of the 5′ and/or 3′ untranslated regionassociated with the genomic nucleic acid sequence; insertion of aheterologous 5′ and/or 3′ untranslated region; and modification of apolyadenylation site. In some embodiments, the non-genomic nucleic acidmolecule is a cDNA.

In some embodiments, the disclosure teaches nucleic acid molecules thatencode insecticidal proteins taught herein, as well as nucleic acidmolecules that encode proteins taught herein that have had an amino acidsubstitution, deletion, insertion, and fragments thereof andcombinations thereof.

Also provided are nucleic acid molecules that encode transcriptionand/or translation products that are subsequently spliced to ultimatelyproduce functional insecticidal proteins. Splicing can be accomplishedin vitro or in vivo, and can involve cis- or trans-splicing. Thesubstrate for splicing can be polynucleotides (e.g., RNA transcripts) orpolypeptides. An example of cis-splicing of a polynucleotide is where anintron inserted into a coding sequence is removed and the two flankingexon regions are spliced to generate an insecticidal protein encodingsequence. An example of trans-splicing would be where a polynucleotideis encrypted by separating the coding sequence into two or morefragments that can be separately transcribed and then spliced to formthe full-length pesticidal protein encoding sequence. The use of asplicing enhancer sequence, which can be introduced into a construct,can facilitate splicing either in cis or trans-splicing of polypeptides(U.S. Pat. Nos. 6,365,377 and 6,531,316). Thus, in some embodiments thepolynucleotides do not directly encode a full-length insecticidalprotein, but rather encode a fragment or fragments of same.

Nucleic acid molecules that are fragments of the aforementionedsequences encoding insecticidal proteins are also encompassed by theembodiments. “Fragment” as used herein refers to a portion of thenucleic acid sequence encoding an insecticidal protein. A fragment of anucleic acid sequence may encode a biologically active portion of aprotein or it may be a fragment that can be used as a hybridizationprobe or PCR primer using methods disclosed herein. Nucleic acidmolecules that are fragments of a nucleic acid sequence comprise atleast about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280,290, 300, or more contiguous nucleotides, or up to the number ofnucleotides present in a full-length nucleic acid sequence encoding aninsecticidal protein taught herein. “Contiguous nucleotides” is usedherein to refer to nucleotide residues that are immediately adjacent toone another. Fragments of the nucleic acid sequences of the embodimentswill encode protein fragments that retain the biological activity of theinsecticidal protein. In some embodiments, a fragment of a nucleic acidsequence will encode at least about 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,240, 250, 260, 270, 280, 290, 300, or more contiguous amino acids, or upto the total number of amino acids present in a full-length insecticidalprotein taught herein. In some embodiments, the fragment is anN-terminal and/or a C-terminal truncation of at least about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more amino acids from theN-terminus and/or C-terminus relative to an insecticidal protein taughtherein, e.g., by proteolysis, insertion of a start codon, deletion ofthe codons encoding the deleted amino acids with the concomitantinsertion of a stop codon or by insertion of a stop codon in the codingsequence.

In some embodiments, an insecticidal protein is encoded by a nucleicacid sequence sufficiently similar to the nucleic acid sequence of SEQID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ IDNO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29,SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO:39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ IDNO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67,SEQ ID NO: 69, or SEQ ID NO: 71. “Sufficiently similar” is used hereinto refer to an amino acid or nucleic acid sequence that has at leastabout 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence similaritycompared to a reference sequence using one of the alignment programsdescribed herein, or known to one of skill in the art, using standardparameters.

In some embodiments, an insecticidal protein is encoded by a nucleicacid sequence that has sufficient sequence identity to the nucleic acidsequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17,SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO:27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ IDNO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55,SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO:65, SEQ ID NO: 67, SEQ ID NO: 69, or SEQ ID NO: 71. “Sufficient sequenceidentity” is used herein to refer to an amino acid or nucleic acidsequence that has at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity compared to a reference sequence using one of thealignment programs described herein, or known to one of skill in theart, using standard parameters.

Percent Identity Calculations

One of skill in the art will recognize that the aforementioned valuescan be appropriately adjusted to determine corresponding homology oridentity of proteins encoded by two nucleic acid sequences by takinginto account codon degeneracy, amino acid similarity, reading framepositioning, and the like. In some embodiments the sequence homology isagainst the full length sequence of the polynucleotide encoding aprotein. In some embodiments, the sequence identity is calculated usingClustalW algorithm in the ALIGNX® module of the Vector NTI® ProgramSuite (Invitrogen Corporation, Carlsbad, Calif.) with all defaultparameters. In some embodiments the sequence identity is across theentire length of polypeptide calculated using ClustalW algorithm in theALIGNX module of the Vector NTI Program Suite (Invitrogen Corporation,Carlsbad, Calif.) with all default parameters.

To determine the percent identity of two amino acid sequences, or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes. The percent identity between the two sequences is a functionof the number of identical positions shared by the sequences (i.e.,percent identity=number of identical positions/total number of positions(e.g., overlapping positions)×100). In one embodiment, the two sequencesare the same length. In another embodiment, the comparison is across theentirety of the reference sequence. The percent identity between twosequences can be determined using techniques similar to those describedbelow, with or without allowing gaps. In calculating percent identity,typically exact matches are counted.

The determination of percent identity between two sequences (nucleicacid or amino acid) can be accomplished using a mathematical algorithm.A non-limiting example of a mathematical algorithm utilized for thecomparison of two sequences is the algorithm of Karlin and Altschul,(1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin andAltschul, (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such analgorithm is incorporated into the BLASTN and BLASTX programs ofAltschul, et al., (1990) J. Mol. Biol. 215:403. BLAST nucleotidesearches can be performed with the BLASTN program, score=100,wordlength=12, to obtain nucleic acid sequences homologous to pesticidalnucleic acid molecules of the embodiments. BLAST protein searches can beperformed with the BLASTX program, score=50, wordlength=3, to obtainamino acid sequences homologous to pesticidal protein molecules of theembodiments. To obtain gapped alignments for comparison purposes, GappedBLAST (in BLAST 2.0) can be utilized as described in Altschul, et al.,(1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be usedto perform an iterated search that detects distant relationships betweenmolecules. See, Altschul, et al., (1997) supra. When utilizing BLAST,Gapped BLAST, and PSI-Blast programs, the default parameters of therespective programs (e.g., BLASTX and BLASTN) can be used. Alignment mayalso be performed manually by inspection.

Another non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the ClustalW algorithm (Higgins, et al.,(1994) Nucleic Acids Res. 22:4673-4680). ClustalW compares sequences andaligns the entirety of the amino acid or DNA sequence, and thus canprovide data about the sequence conservation of the entire amino acidsequence. The ClustalW algorithm is used in several commerciallyavailable DNA/amino acid analysis software packages, such as the ALIGNX®module of the Vector NTI® Program Suite (Invitrogen Corporation,Carlsbad, Calif.). After alignment of amino acid sequences withClustalW, the percent amino acid identity can be assessed. Anon-limiting example of a software program useful for analysis ofClustalW alignments is GENEDOC™. GENE-DOC™ (Karl Nicholas) allowsassessment of amino acid (or DNA) similarity and identity betweenmultiple proteins. Another non-limiting example of a mathematicalalgorithm utilized for the comparison of sequences is the algorithm ofMyers and Miller, (1988) CABIOS 4:11-17. Such an algorithm isincorporated into the ALIGN program (version 2.0), which is part of theGCG Wisconsin Genetics Software Package, Version 10 (available fromAccelrys, Inc., 9685 Scranton Rd., San Diego, Calif., USA). Whenutilizing the ALIGN program for comparing amino acid sequences, a PAM120weight residue table, a gap length penalty of 12, and a gap penalty of 4can be used.

Another non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the algorithm of Needleman and Wunsch,(1970) J. Mol. Biol. 48(3):443-453, used GAP Version 10 software todetermine sequence identity or similarity using the following defaultparameters: % identity and % similarity for a nucleic acid sequenceusing GAP Weight of 50 and Length Weight of 3; % identity or %similarity for an amino acid sequence using GAP weight of 8 and lengthweight of 2, and the BLOSUM62 scoring program. Equivalent programs mayalso be used. Thus, any sequence comparison program that, for any twosequences in question, generates an alignment having identicalnucleotide or amino acid residue matches and calculates a percentsequence identity can be used.

Nucleic Acid Molecule Variants

The disclosure provides nucleic acid molecules encoding insecticidalprotein variants. “Variants” of encoding nucleic acid sequences mayinclude those sequences that encode insecticidal proteins disclosedherein, but that differ conservatively, because of the degeneracy of thegenetic code as well as those that are sufficiently identical asdiscussed above. Naturally occurring allelic variants can be identifiedwith the use of well-known molecular biology techniques, such aspolymerase chain reaction (PCR) and hybridization techniques as outlinedbelow. Variant nucleic acid sequences also include synthetically derivednucleic acid sequences that have been generated, for example, by usingsite-directed mutagenesis, but which still encode the disclosedinsecticidal proteins.

The present disclosure provides isolated or recombinant polynucleotidesthat encode any of the insecticidal proteins disclosed herein. Thosehaving ordinary skill in the art will readily appreciate that due to thedegeneracy of the genetic code, a multitude of nucleotide sequencesencoding proteins of the present disclosure exist. Table A is a codontable that provides the synonymous codons for each amino acid. Forexample, the codons AGA, AGG, CGA, CGC, CGG, and CGU all encode theamino acid arginine. Thus, at every position in the nucleic acids of thedisclosure where an arginine is specified by a codon, the codon can bealtered to any of the corresponding codons described above withoutaltering the encoded polypeptide. It is understood that U in an RNAsequence corresponds to T in a DNA sequence.

TABLE A Synonymous Codon Table Alanine Ala GCA, GCC, GCG, GCU CysteineCys UGC, UGU Aspartic Acid Asp GAC, GAU Glutamic Acid Glu GAA, GAGPhenylalanine Phe UUC, UUU Glycine Gly GGA, GGC, GGG, GGU Histidine HisCAC, CAU Isoleucine Ile AUA, AUC, AUU Lysine Lys AAA, AAG Leucine LeuUUA, UUG, CUA, CUC, CUG, CUU Methionine Met AUG Asparagine Asn AAC, AAUProline Pro CCA, CCC, CCG, CCU Glutamine Gln CAA, CAG Arginine Arg AGA,AGG, CGA, CGC, CGG, CGU Serine Ser AGC, AGU, UCA, UCC, UCG, UCUThreonine Thr ACA, ACC, ACG, ACU Valine Val GUA, GUC, GUG, UU TryptophanTrp UGG Tyrosine Tyr UAC, UAU

The skilled artisan will further appreciate that changes can beintroduced by mutation of the nucleic acid sequences thereby leading tochanges in the amino acid sequence of the encoded proteins, withoutaltering the biological activity of the proteins. Thus, variant nucleicacid molecules can be created by introducing one or more nucleotidesubstitutions, additions, and/or deletions into the correspondingnucleic acid sequence disclosed herein, such that one or more amino acidsubstitutions, additions or deletions are introduced into the encodedprotein. Mutations can be introduced by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis. Such variantnucleic acid sequences are also encompassed by the present disclosure.

Alternatively, variant nucleic acid sequences can be made by introducingmutations randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forability to confer pesticidal activity to identify mutants that retainactivity. Following mutagenesis, the encoded protein can be expressedrecombinantly, and the activity of the protein can be determined usingstandard assay techniques.

The polynucleotides of the disclosure and fragments thereof areoptionally used as substrates for a variety of recombination andrecursive recombination reactions, in addition to standard cloningmethods as set forth in, e.g., Ausubel, Berger and Sambrook, i.e., toproduce additional pesticidal protein homologues and fragments thereofwith desired properties. A variety of such reactions are known. Methodsfor producing a variant of any nucleic acid listed herein comprisingrecursively recombining such polynucleotide with a second (or more)polynucleotide, thus forming a library of variant polynucleotides arealso embodiments of the disclosure, as are the libraries produced, thecells comprising the libraries, and any recombinant polynucleotideproduced by such methods.

A variety of diversity generating protocols, including nucleic acidrecursive recombination protocols are available and fully described inthe art. The procedures can be used separately, and/or in combination toproduce one or more variants of a nucleic acid or set of nucleic acids,as well as variants of encoded proteins. Individually and collectively,these procedures provide robust, widely applicable ways of generatingdiversified nucleic acids and sets of nucleic acids (including, e.g.,nucleic acid libraries) useful, e.g., for the engineering or rapidevolution of nucleic acids, proteins, pathways, cells and/or organismswith new and/or improved characteristics.

While distinctions and classifications are made in the course of theensuing discussion for clarity, it will be appreciated that thetechniques are often not mutually exclusive. Indeed, the various methodscan be used singly or in combination, in parallel or in series, toaccess diverse sequence variants.

The result of any of the diversity generating procedures describedherein can be the generation of one or more nucleic acids, which can beselected or screened for nucleic acids with or which confer desirableproperties or that encode proteins with or which confer desirableproperties. Following diversification by one or more of the methodsherein or otherwise available to one of skill, any nucleic acids thatare produced can be selected for a desired activity or property, e.g.pesticidal activity. This can include identifying any activity that canbe detected, for example, in an automated or automatable format, by anyof the assays in the art, see, e.g., discussion of screening ofinsecticidal activity, infra. A variety of related (or even unrelated)properties can be evaluated, in serial or in parallel, at the discretionof the practitioner.

Descriptions of a variety of diversity generating procedures forgenerating modified nucleic acid sequences, e.g., those coding forproteins having pesticidal activity or fragments thereof, are found inthe following publications and the references cited therein: Soong, etal., (2000) Nat. Genet. 25(4):436-439; Stemmer, et al., (1999) TumorTargeting 4: 1-4; Ness, et al., (1999) Nat. Biotechnol. 17:893-896;Chang, et al., (1999) Nat. Biotechnol. 17:793-797; Minshull and Stemmer,(1999) Curr. Opin. Chem. Biol. 3:284-290; Christians, et al., (1999)Nat. Biotechnol. 17:259-264; Crameri, et al., (1998) Nature 391:288-291;Crameri, et al., (1997) Nat. Biotechnol. 15:436-438; Zhang, et al.,(1997) PNAS USA 94:4504-4509; Patten, et al., (1997) Curr. Opin.Biotechnol. 8:724-733; Crameri, et al., (1996) Nat. Med. 2:100-103;Crameri, et al., (1996) Nat. Biotechnol. 14:315-319; Gates, et al.,(1996) J. Mol. Biol. 255:373-386; Stemmer, (1996) “Sexual PCR andAssembly PCR” In: The Encyclopedia of Molecular Biology. VCH Publishers,New York. pp. 447-457; Crameri and Stemmer, (1995) BioTechniques 18:194-195; Stemmer, et al., (1995) Gene, 164:49-53; Stemmer, (1995)Science 270: 1510; Stemmer, (1995) Biotechnology 13:549-553; Stemmer,(1994) Nature 370:389-391 and Stemmer, (1994) PNAS USA 91:10747-10751.

Mutational methods of generating diversity include, for example,site-directed mutagenesis (Ling, et al., (1997) Anal Biochem 254(2):157-178; Dale, et al., (1996) Methods Mol. Biol. 57:369-374; Smith,(1985) Ann. Rev. Genet. 19:423-462; Botstein and Shortle, (1985) Science229:1193-1201; Carter, (1986) Biochem. J. 237: 1-7 and Kunkel, (1987)“The efficiency of oligonucleotide directed mutagenesis” in NucleicAcids & Molecular Biology (Eckstein and Lilley, eds., Springer Verlag,Berlin)); mutagenesis using uracil containing templates (Kunkel, (1985)PNAS USA 82:488-492; Kunkel, et al., (1987) Methods Enzymol. 154:367-382and Bass, et al., (1988) Science 242:240-245); oligonucleotide-directedmutagenesis (Zoller and Smith, (1983) Methods Enzymol. 100:468-500;Zoller and Smith, (1987) Methods Enzymol. 154:329-350 (1987); Zoller andSmith, (1982) Nucleic Acids Res. 10:6487-6500),phosphorothioate-modified DNA mutagenesis (Taylor, et al., (1985)Nucleic Acids Res. 13:8749-8764; Taylor, et al., (1985) Nucleic AcidsRes. 13:8765-8787 (1985); Nakamaye and Eckstein, (1986) Nucleic AcidsRes. 14:9679-9698; Sayers, et al., (1988) Nucleic Acids Res. 16:791-802and Sayers, et al., (1988) Nucleic Acids Res. 16:803-814); mutagenesisusing gapped duplex DNA (Kramer, et al., (1984) Nucleic Acids Res.12:9441-9456; Kramer and Fritz, (1987) Methods Enzymol. 154:350-367;Kramer, et al., (1988) Nucleic Acids Res. 16:7207 and Fritz, et al.,(1988) Nucleic Acids Res. 16:6987-6999).

Additional suitable methods include point mismatch repair (Kramer, etal., (1984) Cell 38:879-887), mutagenesis using repair-deficient hoststrains (Carter, et al., (1985) Nucleic Acids Res. 13:4431-4443 andCarter, (1987) Methods in Enzymol. 154:382-403), deletion mutagenesis(Eghtedarzadeh and Henikoff, (1986) Nucleic Acids Res. 14: 5115),restriction-selection and restriction-purification (Wells, et al.,(1986) Phil. Trans. R. Soc. Lond. A317:415-423), mutagenesis by totalgene synthesis (Nambiar, et al., (1984) Science 223:1299-1301; Sakamarand Khorana, (1988) Nucleic Acids Res. 14:6361-6372; Wells, et al.,(1985) Gene 34:315-323 and Grundstriim, et al., (1985) Nucleic AcidsRes. 13:3305-3316), double-strand break repair (Mandecki, (1986) PNASUSA, 83:7177-7181 and Arnold, (1993) Curr. Opin. Biotech. 4:450-455).Additional details on many of the above methods can be found in MethodsEnzymol Volume 154, which also describes useful controls fortrouble-shooting problems with various mutagenesis methods.

Additional details regarding various diversity generating methods can befound in the following US Patents, PCT Publications, and Applicationsand EPO publications: U.S. Pat. Nos. 5,723,323, 5,763,192, 5,814,476,5,817,483, 5,824,514, 5,976,862, 5,605,793, 5,811,238, 5,830,721,5,834,252, 5,837,458, WO 1995/22625, WO 1996/33207, WO 1997/20078, WO1997/35966, WO 1999/41402, WO 1999/41383, WO 1999/41369, WO 1999/41368,EP 752008, EP 0932670, WO 1999/23107, WO 1999/21979, WO 1998/31837, WO1998/27230, WO 1998/27230, WO 2000/00632, WO 2000/09679, WO 1998/42832,WO 1999/29902, WO 1998/41653, WO 1998/41622, WO 1998/42727, WO2000/18906, WO 2000/04190, WO 2000/42561, WO 2000/42559, WO 2000/42560,and WO 2001/23401.

Nucleic Acid Molecule Probes to Find Related Nucleic Acids

The nucleotide sequences of the embodiments can also be used to isolatecorresponding sequences from other organisms, particularly otherbacteria, particularly a Pseudomonas species. In this manner, methodssuch as PCR, hybridization, and the like can be used to identify suchsequences based on their sequence homology to the sequences set forthherein.

Sequences that are selected based on their sequence identity to theentire sequences set forth herein or to fragments thereof areencompassed by the embodiments. Such sequences include sequences thatare orthologs of the disclosed sequences. The term “orthologs” refers togenes derived from a common ancestral gene and which are found indifferent species as a result of speciation. Genes found in differentspecies are considered orthologs when their nucleotide sequences and/ortheir encoded protein sequences share substantial identity as definedelsewhere herein. Functions of orthologs are often highly conservedamong species.

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any organism of interest. Methods fordesigning PCR primers and PCR cloning are generally known in the art andare disclosed in Sambrook, et al., (1989) Molecular Cloning: ALaboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.), hereinafter “Sambrook”. See also, Innis, et al., eds.(1990) PCR Protocols: A Guide to Methods and Applications (AcademicPress, New York); Innis and Gelfand, eds. (1995) PCR Strategies(Academic Press, New York); and Innis and Gelfand, eds. (1999) PCRMethods Manual (Academic Press, New York). Known methods of PCR include,but are not limited to, methods using paired primers, nested primers,single specific primers, degenerate primers, gene-specific primers,vector specific primers, partially-mismatched primers, and the like.

To identify insecticidal proteins of the disclosure from bacterialcollections, the bacterial cell lysates can be screened with antibodiesgenerated against a taught protein using Western blotting and/or ELISAmethods. This type of assay can be performed in a high throughputfashion. Positive samples can be further analyzed by various techniquessuch as antibody based protein purification and identification. Methodsof generating antibodies are well known in the art as discussed infra.

Alternatively, mass spectrometry based protein identification methodscan be used to identify homologs of the taught proteins using protocolsin the literature (Scott Patterson, (1998), 10.22, 1-24, CurrentProtocol in Molecular Biology published by John Wiley & Son Inc).Specifically, LC-MS/MS based protein identification methods can be usedto associate the MS data of given cell lysate or desired molecularweight enriched samples (excised from SDS-PAGE gel of relevant molecularweight bands to proteins taught herein) with sequence information of thetaught proteins and homologs. Any match in peptide sequences indicatesthe potential of having the homologs in the samples. Additionaltechniques (protein purification and molecular biology) can be used toisolate the protein and identify the sequences of the homologs.

In hybridization methods, all or part of the pesticidal nucleic acidsequence can be used to screen cDNA or genomic libraries. Methods forconstruction of such cDNA and genomic libraries are generally known inthe art and are disclosed in Sambrook and Russell, (2001), supra. Theso-called hybridization probes may be genomic DNA fragments, cDNAfragments, RNA fragments or other oligonucleotides and may be labeledwith a detectable group such as 32P or any other detectable marker, suchas other radioisotopes, a fluorescent compound, an enzyme or an enzymecofactor. Probes for hybridization can be made by labeling syntheticoligonucleotides based on the known peptide-encoding nucleic acidsequence disclosed herein. Degenerate primers designed on the basis ofconserved nucleotides or amino acid residues in the nucleic acidsequence or encoded amino acid sequence can additionally be used. Theprobe typically comprises a region of nucleic acid sequence thathybridizes under stringent conditions to at least about 12, at leastabout 25, at least about 50, 75, 100, 125, 150, 175 or 200 consecutivenucleotides of nucleic acid sequence encoding a protein of thedisclosure or a fragment or variant thereof. Methods for the preparationof probes for hybridization are generally known in the art and aredisclosed in Sambrook and Russell, (2001), supra, herein incorporated byreference.

For example, an entire nucleic acid sequence, encoding an insecticidalprotein taught herein, or one or more portions thereof may be used as aprobe capable of specifically hybridizing to corresponding nucleic acidsequences encoding like sequences and messenger RNAs. To achievespecific hybridization under a variety of conditions, such probesinclude sequences that are unique and are preferably at least about 10nucleotides in length or at least about 20 nucleotides in length. Suchprobes may be used to amplify corresponding pesticidal sequences from achosen organism by PCR. This technique may be used to isolate additionalcoding sequences from a desired organism or as a diagnostic assay todetermine the presence of coding sequences in an organism. Hybridizationtechniques include hybridization screening of plated DNA libraries(either plaques or colonies; see, for example, Sambrook, et al., (1989)Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.).

Hybridization of such sequences may be carried out under stringentconditions. “Stringent conditions” or “stringent hybridizationconditions” is used herein to refer to conditions under which a probewill hybridize to its target sequence to a detectably greater degreethan to other sequences (e.g., at least 2-fold over background).Stringent conditions are sequence dependent and will be different indifferent circumstances. By controlling the stringency of thehybridization and/or washing conditions, target sequences that are 100%complementary to the probe can be identified (homologous probing).Alternatively, stringency conditions can be adjusted to allow somemismatching in sequences so that lower degrees of similarity aredetected (heterologous probing). Generally, a probe is less than about1000 nucleotides in length, preferably less than 500 nucleotides inlength.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1× SSC at 55 to 60°. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffersmay comprise about 0.1% to about 1% SDS. Duration of hybridization isgenerally less than about 24 hours, usually about 4 to about 12 hours.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the Tm can be approximated from theequation of Meinkoth and Wahl, (1984) Anal. Biochem. 138:267-284:Tm=81.5° C.+16.6(log M)+0.41(% GC)−0.61(% form)−500/L; where M is themolarity of monovalent cations, % GC is the percentage of guanosine andcytosine nucleotides in the DNA, % form is the percentage of formamidein the hybridization solution, and L is the length of the hybrid in basepairs. The Tm is the temperature (under defined ionic strength and pH)at which 50% of a complementary target sequence hybridizes to aperfectly matched probe. Tm is reduced by about 1° C. for each 1% ofmismatching; thus, Tm, hybridization, and/or wash conditions can beadjusted to hybridize to sequences of the desired identity. For example,if sequences with 90% identity are sought, the Tm can be decreased 10°C. Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (Tm) for the specific sequence and itscomplement at a defined ionic strength and pH. However, severelystringent conditions can utilize a hybridization and/or wash at 1, 2, 3or 4° C. lower than the thermal melting point (Tm); moderately stringentconditions can utilize a hybridization and/or wash at 6, 7, 8, 9 or 10°C. lower than the thermal melting point (Tm); low stringency conditionscan utilize a hybridization and/or wash at 11, 12, 13, 14, 15 or 20° C.lower than the thermal melting point (Tm). Using the equation,hybridization and wash compositions, and desired Tm, those of ordinaryskill will understand that variations in the stringency of hybridizationand/or wash solutions are inherently described. If the desired degree ofmismatching results in a Tm of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen, (1993)Laboratory Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, N.Y.); and Ausubel, et al., eds. (1995) Current Protocols inMolecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience,New York). See, Sambrook, et al., (1989) Molecular Cloning: A LaboratoryManual (2nd ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.).

Newly Discovered Insecticidal Proteins, Variants, and Fragments Thereof

Novel insecticidal proteins are disclosed herein, along with variants ofsaid proteins, and fragments thereof. The terms “proteins” and“polypeptides” are in some instances used interchangeably, as it isunderstood in the art that the separation between the two terms canmerely depend upon the number of amino acid sequences. The insecticidalproteins of the disclosure demonstrate insecticidal or pesticidalactivity against one or more insects or pests.

In some embodiments, an insecticidal protein is sufficiently homologousto the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ IDNO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44,SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO:54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ IDNO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, or SEQ ID NO: 72.“Sufficiently homologous” is used herein to refer to an amino acid ornucleic acid sequence that has at least about 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% sequence homology compared to a referencesequence using one of the alignment programs described herein, or knownto one of skill in the art, using standard parameters.

In some embodiments, an insecticidal protein has sufficient sequenceidentity to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24,SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO:34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ IDNO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62,SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, or SEQ IDNO: 72. “Sufficient sequence identity” is used herein to refer to anamino acid or nucleic acid sequence that has at least about 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to areference sequence using one of the alignment programs described herein,or known to one of skill in the art, using standard parameters.

In some embodiments, the disclosure provides for an amino acid sequenceof: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO:10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ IDNO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38,SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO:48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ IDNO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQID NO: 68, SEQ ID NO: 70, or SEQ ID NO: 72, which are encoded by anucleic acid sequence of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25,SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO:35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ IDNO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63,SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, or SEQ ID NO: 71.

As used herein, the terms “protein,” “peptide” or “polypeptide” includesany molecule that comprises five or more amino acids. It is well knownin the art that protein, peptide, or polypeptide molecules may undergomodification, including post-translational modifications, such as, butnot limited to, disulfide bond formation, glycosylation, phosphorylationor oligomerization. Thus, as used herein, the terms “protein,” “peptidemolecule” or “polypeptide” includes any protein that is modified by anybiological or non-biological process.

A “recombinant protein” is used herein to refer to a protein that is nolonger in its natural environment, for example in vitro or in arecombinant bacterial or plant host cell. An insecticidal protein thatis substantially free of cellular material includes preparations ofprotein having less than about 30%, 20%, 10% or 5% (by dry weight) ofnon-pesticidal protein (also referred to herein as a “contaminatingprotein”).

“Fragments” or “biologically active portions” include protein fragmentscomprising amino acid sequences sufficiently identical to a proteintaught herein and that exhibit insecticidal activity.

Thus, the disclosure contemplates fragments of the amino acid sequencesset forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18,SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO:28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ IDNO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56,SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO:66, SEQ ID NO: 68, SEQ ID NO: 70, or SEQ ID NO: 72.

In some embodiments, the protein fragment is an N-terminal and/or aC-terminal truncation of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14 or more amino acids from the N-terminus and/or C-terminusrelative to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18,SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO:28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ IDNO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56,SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO:66, SEQ ID NO: 68, SEQ ID NO: 70, or SEQ ID NO: 72, e.g., byproteolysis, by insertion of a start codon, by deletion of the codonsencoding the deleted amino acids and concomitant insertion of a startcodon, and/or insertion of a stop codon.

“Variants” as used herein refers to proteins or polypeptides having anamino acid sequence that is at least about 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to the parental amino acid sequence.

The term “about” as used herein with respect to % sequence identity of anucleic acid or amino acid means up to and including ±1.0% in 0.1%increments. For example “about 90%” sequence identity includes 89.0%,89.1%, 89.2%, 89.3%, 89.4%, 89.5%, 89.6%, 89.7%, 89.8%, 89.9%, 90%,90.1%, 90.2%, 90.3%, 90.4%, 90.5%, 90.6%, 90.7%, 90.8%, 90.9%, and 91%.If not used in the context of % sequence identity, then “about”means±10%.

In some embodiments, an insecticidal protein has at least about 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity across the entirelength of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ IDNO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34,SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO:44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ IDNO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, or SEQ ID NO:72.

In some embodiments, the insecticidal proteins have a modified physicalproperty. As used herein, the term “physical property” refers to anyparameter suitable for describing the physical-chemical characteristicsof a protein. As used herein, “physical property of interest” and“property of interest” are used interchangeably to refer to physicalproperties of proteins that are being investigated and/or modified.Examples of physical properties include, but are not limited to: netsurface charge and charge distribution on the protein surface, nethydrophobicity and hydrophobic residue distribution on the proteinsurface, surface charge density, surface hydrophobicity density, totalcount of surface ionizable groups, surface tension, protein size and itsdistribution in solution, melting temperature, heat capacity, and secondvirial coefficient. Examples of physical properties also include, butare not limited to: solubility, folding, stability, and digestibility.In some embodiments, the taught insecticidal protein has increaseddigestibility of proteolytic fragments in an insect gut. Models fordigestion by simulated gastric fluids are known to one skilled in theart (Fuchs, R. L. and J. D. Astwood. Food Technology 50: 83-88, 1996;Astwood, J. D., et al Nature Biotechnology 14: 1269-1273, 1996; Fu T Jet al J. Agric. Food Chem. 50: 7154-7160, 2002).

In some embodiments, variants include polypeptides that differ in aminoacid sequence due to mutagenesis. Variant proteins encompassed by thedisclosure are biologically active, that is they continue to possess thedesired biological activity (i.e. pesticidal activity) of the nativeprotein. In some embodiments, the variant will have at least about 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of insecticidal activityof the native protein. In some embodiments, the variants may haveimproved activity over the native protein.

Bacterial genes quite often possess multiple methionine initiationcodons in proximity to the start of the open reading frame. Often,translation initiation at one or more of these start codons will lead togeneration of a functional protein. These start codons can include ATGcodons. However, bacteria such as Bacillus sp. also recognize the codonGTG as a start codon, and proteins that initiate translation at GTGcodons contain a methionine at the first amino acid. On rare occasions,translation in bacterial systems can initiate at a TTG codon, though inthis event the TTG encodes a methionine. Furthermore, it is not oftendetermined a priori which of these codons are used naturally in thebacterium. Thus, it is understood that use of one of the alternatemethionine codons may also lead to generation of pesticidal proteins.These pesticidal proteins are encompassed in the present disclosure andmay be used in the methods of the present disclosure. It will beunderstood that, when expressed in plants, it will be necessary to alterthe alternate start codon to ATG for proper translation.

In another aspect, the insecticidal protein may be expressed as aprecursor protein with an intervening sequence that catalyzes multistep,post translational protein splicing. Protein splicing involves theexcision of an intervening sequence from a polypeptide with theconcomitant joining of the flanking sequences to yield a new polypeptide(Chong, et al., (1996) J. Biol. Chem., 271:22159-22168). Thisintervening sequence or protein splicing element, referred to asinteins, which catalyze their own excision through three coordinatedreactions at the N-terminal and C-terminal splice junctions: an acylrearrangement of the N-terminal cysteine or serine; atransesterification reaction between the two termini to form a branchedester or thioester intermediate and peptide bond cleavage coupled tocyclization of the intein C-terminal asparagine to free the intein(Evans, et al., (2000) J. Biol. Chem., 275:9091-9094. The elucidation ofthe mechanism of protein splicing has led to a number of intein-basedapplications (Comb, et al., U.S. Pat. No. 5,496,714; Comb, et al., U.S.Pat. No. 5,834,247; Camarero and Muir, (1999) J. Amer. Chem. Soc.121:5597-5598; Chong, et al., (1997) Gene 192:271-281, Chong, et al.,(1998) Nucleic Acids Res. 26:5109-5115; Chong, et al., (1998) J. Biol.Chem. 273:10567-10577; Cotton, et al., (1999) J. Am. Chem. Soc.121:1100-1101; Evans, et al., (1999) J. Biol. Chem. 274:18359-18363;Evans, et al., (1999) J. Biol. Chem. 274:3923-3926; Evans, et al.,(1998) Protein Sci. 7:2256-2264; Evans, et al., (2000) J. Biol. Chem.275:9091-9094; Iwai and Pluckthun, (1999) FEBS Lett. 459:166-172;Mathys, et al., (1999) Gene 231:1-13; Mills, et al., (1998) Proc. Natl.Acad. Sci. USA 95:3543-3548; Muir, et al., (1998) Proc. Natl. Acad. Sci.USA 95:6705-6710; Otomo, et al., (1999) Biochemistry 38:16040-16044;Otomo, et al., (1999) J. Biolmol. NMR 14:105-114; Scott, et al., (1999)Proc. Natl. Acad. Sci. USA 96: 13638-13643; Severinov and Muir, (1998)J. Biol. Chem. 273:16205-16209; Shingledecker, et al., (1998) Gene 207:187-195; Southworth, et al., (1998) EMBO J. 17:918-926; Southworth, etal., (1999) Biotechniques 27: 110-120; Wood, et al., (1999) Nat.Biotechnol. 17:889-892; Wu, et al., (1998a) Proc. Natl. Acad. Sci. USA95:9226-9231; Wu, et al., (1998b) Biochim. Biophys. Acta 1387:422-432;Xu, et al., (1999) Proc. Natl. Acad. Sci. USA 96:388-393; Yamazaki, etal., (1998) J. Am. Chem. Soc., 120:5591-5592). For the application ofinteins in plant transgenes, see, Yang, et al., (Transgene Res.15:583-593 (2006)) and Evans, et al., (Annu. Rev. Plant Biol. 56:375-392(2005)).

In another aspect, the insecticidal protein may be encoded by twoseparate genes where the intein of the precursor protein comes from thetwo genes, referred to as a split intein, and the two portions of theprecursor are joined by a peptide bond formation. This peptide bondformation is accomplished by intein-mediated trans-splicing. For thispurpose, a first and a second expression cassette comprising the twoseparate genes further code for inteins capable of mediating proteintrans-splicing. By trans-splicing, the proteins and polypeptides encodedby the first and second fragments may be linked by peptide bondformation. Trans-splicing inteins may be selected from the nucleolar andorganelle genomes of different organisms including eukaryotes,archaebacteria and eubacteria. Inteins that may be used are listed atneb.com/neb/inteins.html, which can be accessed on the worldwide webusing the “www” prefix. The nucleotide sequence coding for an intein maybe split into a 5′ and a 3′ part that code for the 5′ and the 3′ part ofthe intein, respectively. Sequence portions not necessary for inteinsplicing (e.g. homing endonuclease domain) may be deleted. The inteincoding sequence is split such that the 5′ and the 3′ parts are capableof trans-splicing. For selecting a suitable splitting site of the inteincoding sequence, the considerations published by Southworth, et al.,(1998) EMBO J. 17:918-926 may be followed. In constructing the first andthe second expression cassette, the 5′ intein coding sequence is linkedto the 3′ end of the first fragment coding for the N-terminal part ofpolypeptide and the 3′ intein coding sequence is linked to the 5′ end ofthe second fragment coding for the C-terminal part of the polypeptide.

In general, the trans-splicing partners can be designed using any splitintein, including any naturally occurring or artificially split intein.Several naturally occurring split inteins are known, for example: thesplit intein of the DnaE gene of Synechocystis sp. PCC6803 (see, Wu, etal., (1998) Proc. Natl. Acad. Sci. USA. 95(16):9226-31 and Evans, etal., (2000) J. Biol. Chem. 275(13):9091-4 and of the DnaE gene fromNostoc punctiforme (see, Iwai, et al., (2006) FEBS Lett. 580(7):1853-8). Non-split inteins have been artificially split in thelaboratory to create new split inteins, for example: the artificiallysplit Ssp DnaB intein (see, Wu, et al., (1998) Biochim. Biophys. Acta.1387:422-32) and split See VMA intein (see, Brenzel, et al., (2006)Biochemistry 45(6):1571-8) and an artificially split fungal mini-intein(see, Elleuche, et al., (2007) Biochem. Biophys. Res. Commun.355(3):830-4). Naturally occurring non-split inteins may haveendonuclease or other enzymatic activities that can typically be removedwhen designing an artificially-split split intein. Such mini-inteins orminimized split inteins are well known in the art and are typically lessthan 200 amino acid residues long (see, Wu, et al., (1998) Biochim.Biophys. Acta. 1387: 422-32). Suitable split inteins may have otherpurification enabling polypeptide elements added to their structure,provided that such elements do not inhibit the splicing of the splitintein or are added in a manner that allows them to be removed prior tosplicing. Protein splicing has been reported using proteins thatcomprise bacterial intein-like (BIL) domains (see, Amitai, et al.,(2003) Mol. Microbiol. 47:61-73) and hedgehog (Hog) auto-processingdomains (the latter is combined with inteins when referred to as theHog/intein superfamily or HINT family (see, Dassa, et al., (2004) J.Biol. Chem. 279:32001-7) and domains such as these may also be used toprepare artificially-split inteins. In particular, non-splicing membersof such families may be modified by molecular biology methodologies tointroduce or restore splicing activity in such related species.

The development of recombinant DNA methods has made it possible to studythe effects of sequence transposition on protein folding, structure andfunction. The approach used in creating new sequences resembles that ofnaturally occurring pairs of proteins that are related by linearreorganization of their amino acid sequences (Cunningham, et al., (1979)Proc. Natl. Acad. Sci. U.S.A. 76:3218-3222; Teather and Erfle, (1990) J.Bacteriol. 172:3837-3841; Schimming, et al., (1992) Eur. J. Biochem.204:13-19; Yamiuchi and Minamikawa, (1991) FEBS Lett. 260:127-130;MacGregor, et al., (1996) FEBS Lett. 378:263-266). The first in vitroapplication of this type of rearrangement to proteins was described byGoldenberg and Creighton (J. Mol. Biol. 165:407-413, 1983). In creatinga circular permuted variant, a new N-terminus is selected at an internalsite (breakpoint) of the original sequence, the new sequence having thesame order of amino acids as the original from the breakpoint until itreaches an amino acid that is at or near the original C-terminus. Atthis point the new sequence is joined, either directly or through anadditional portion of sequence (linker), to an amino acid that is at ornear the original N-terminus and the new sequence continues with thesame sequence as the original until it reaches a point that is at ornear the amino acid that was N-terminal to the breakpoint site of theoriginal sequence, this residue forming the new C-terminus of the chain.The length of the amino acid sequence of the linker can be selectedempirically or with guidance from structural information or by using acombination of the two approaches. When no structural information isavailable, a small series of linkers can be prepared for testing using adesign whose length is varied in order to span a range from 0 to 50 Åand whose sequence is chosen in order to be consistent with surfaceexposure (hydrophilicity, Hopp and Woods, (1983) Mol. Immunol.20:483-489; Kyte and Doolittle, (1982) J. Mol. Biol. 157: 105-132;solvent exposed surface area, Lee and Richards, (1971) J. Mol. Biol.55:379-400) and the ability to adopt the necessary conformation withoutderanging the configuration of the pesticidal polypeptide(conformationally flexible; Karplus and Schulz, (1985)Naturwissenschaften 72:212-213). Assuming an average of translation of2.0 to 3.8 Å per residue, this would mean the length to test would bebetween 0 to 30 residues, with 0 to 15 residues being the preferredrange. Exemplary of such an empirical series would be to constructlinkers using a cassette sequence such as Gly-Gly-Gly-Ser repeated ntimes, where n is 1, 2, 3 or 4. Those skilled in the art will recognizethat there are many such sequences that vary in length or compositionthat can serve as linkers with the primary consideration being that theybe neither excessively long nor short (cf., Sandhu, (1992) Critical Rev.Biotech. 12:437-462); if they are too long, entropy effects will likelydestabilize the three-dimensional fold, and may also make foldingkinetically impractical, and if they are too short, they will likelydestabilize the molecule because of torsional or steric strain. Thoseskilled in the analysis of protein structural information will recognizethat using the distance between the chain ends, defined as the distancebetween the c-alpha carbons, can be used to define the length of thesequence to be used or at least to limit the number of possibilitiesthat must be tested in an empirical selection of linkers. They will alsorecognize that it is sometimes the case that the positions of the endsof the polypeptide chain are ill defined in structural models derivedfrom x-ray diffraction or nuclear magnetic resonance spectroscopy data,and that when true, this situation will therefore need to be taken intoaccount in order to properly estimate the length of the linker required.From those residues whose positions are well defined are selected tworesidues that are close in sequence to the chain ends, and the distancebetween their c-alpha carbons is used to calculate an approximate lengthfor a linker between them. Using the calculated length as a guide,linkers with a range of number of residues (calculated using 2 to 3.8 Åper residue) are then selected. These linkers may be composed of theoriginal sequence, shortened or lengthened as necessary, and whenlengthened the additional residues may be chosen to be flexible andhydrophilic as described above; or optionally the original sequence maybe substituted for using a series of linkers, one example being theGly-Gly-Gly-Ser cassette approach mentioned above; or optionally acombination of the original sequence and new sequence having theappropriate total length may be used. Sequences of pesticidalpolypeptides capable of folding to biologically active states can beprepared by appropriate selection of the beginning (amino terminus) andending (carboxyl terminus) positions from within the originalpolypeptide chain while using the linker sequence as described above.Amino and carboxyl termini are selected from within a common stretch ofsequence, referred to as a breakpoint region, using the guidelinesdescribed below. A novel amino acid sequence is thus generated byselecting amino and carboxyl termini from within the same breakpointregion. In many cases the selection of the new termini will be such thatthe original position of the carboxyl terminus immediately preceded thatof the amino terminus. However, those skilled in the art will recognizethat selections of termini anywhere within the region may function, andthat these will effectively lead to either deletions or additions to theamino or carboxyl portions of the new sequence.

It is a central tenet of molecular biology that the primary amino acidsequence of a protein dictates folding to the three-dimensionalstructure necessary for expression of its biological function. Methodsare known to those skilled in the art to obtain and interpret threedimensional structural information using x-ray diffraction of singleprotein Crystals or nuclear magnetic resonance spectroscopy of proteinsolutions. Examples of structural information that are relevant to theidentification of breakpoint regions include the location and type ofprotein secondary structure (alpha and 3-10 helices, parallel andanti-parallel beta sheets, chain reversals and turns, and loops; Kabschand Sander, (1983) Biopolymers 22:2577-2637; the degree of solventexposure of amino acid residues, the extent and type of interactions ofresidues with one another (Chothia, (1984) Ann. Rev. Biochem.53:537-572) and the static and dynamic distribution of conformationsalong the polypeptide chain (Alber and Mathews, (1987) Methods Enzymol.154:511-533). In some cases additional information is known aboutsolvent exposure of residues; one example is a site of posttranslationalattachment of carbohydrate that is necessarily on the surface of theprotein. When experimental structural information is not available or isnot feasible to obtain, methods are also available to analyze theprimary amino acid sequence in order to make predictions of proteintertiary and secondary structure, solvent accessibility and theoccurrence of turns and loops. Biochemical methods are also sometimesapplicable for empirically determining surface exposure when directstructural methods are not feasible; for example, using theidentification of sites of chain scission following limited proteolysisin order to infer surface exposure (Gentile and Salvatore, (1993) Eur.J. Biochem. 218:603-621). Thus using either the experimentally derivedstructural information or predictive methods (e.g., Srinivisan and Rose,(1995) Proteins: Struct., Funct. & Genetics 22:81-99) the parental aminoacid sequence is inspected to classify regions according to whether ornot they are integral to the maintenance of secondary and tertiarystructure. The occurrence of sequences within regions that are known tobe involved in periodic secondary structure (alpha and 3-10 helices,parallel and anti-parallel beta sheets) are regions that should beavoided. Similarly, regions of amino acid sequence that are observed orpredicted to have a low degree of solvent exposure are more likely to bepart of the so-called hydrophobic core of the protein and should also beavoided for selection of amino and carboxyl termini. In contrast, thoseregions that are known or predicted to be in surface turns or loops, andespecially those regions that are known not to be required forbiological activity, are the preferred sites for location of theextremes of the polypeptide chain. Continuous stretches of amino acidsequence that are preferred based on the above criteria are referred toas a breakpoint region. Polynucleotides encoding circular permutedpolypeptides with new N-terminus/C-terminus which contain a linkerregion separating the original C-terminus and N-terminus can be madeessentially following the method described in Mullins, et al., (1994) J.Am. Chem. Soc. 116:5529-5533. Multiple steps of polymerase chainreaction (PCR) amplifications are used to rearrange the DNA sequenceencoding the primary amino acid sequence of the protein. Polynucleotidesencoding circular permuted polypeptides with new N-terminus/C-terminuswhich contain a linker region separating the original C-terminus andN-terminus can be made based on the tandem duplication method describedin Horlick, et al., (1992) Protein Eng. 5:427-431. Polymerase chainreaction (PCR) amplification of the new N-terminus/C-terminus genes isperformed using a tandemly duplicated template DNA.

Fusion Proteins Comprising the Novel Insecticidal Proteins

In another aspect, fusion proteins are provided that include within itsamino acid sequence a sequence selected from the group consisting of SEQID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20,SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO:30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ IDNO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58,SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO:68, SEQ ID NO: 70, and SEQ ID NO: 72, and active fragments thereof.

Methods for design and construction of fusion proteins (andpolynucleotides encoding same) are known to those of skill in the art.Polynucleotides encoding an insecticidal protein may be fused to signalsequences which will direct the localization of the polypeptide toparticular compartments of a prokaryotic or eukaryotic cell and/ordirect the secretion of the polypeptide from a prokaryotic or eukaryoticcell. For example, in E. coli, one may wish to direct the expression ofthe protein to the periplasmic space.

Examples of signal sequences or proteins (or fragments thereof) to whichthe insecticidal polypeptide may be fused, in order to direct theexpression of the polypeptide to the periplasmic space of bacteriainclude, but are not limited to: the pelB signal sequence, the maltosebinding protein (MBP) signal sequence, MBP, the ompA signal sequence,the signal sequence of the periplasmic E. coli heat labile enterotoxinB-subunit, and the signal sequence of alkaline phosphatase.

Several vectors are commercially available for the construction offusion proteins, which will direct the localization of a protein, suchas the pMAL series of vectors (pMAL-p series) available from New EnglandBiolabs® (240 County Road, Ipswich, Mass. 01938-2723). In a specificembodiment, the polypeptide may be fused to the pelB pectate lyasesignal sequence to increase the efficiency of expression andpurification of such polypeptides in Gram-negative bacteria (see, U.S.Pat. Nos. 5,576,195 and 5,846,818). Plant plastid transitpeptide/polypeptide fusions are well known in the art (see, U.S. Pat.No. 7,193,133). Apoplast transit peptides such as rice or barleyalpha-amylase secretion signal are also well known in the art. Theplastid transit peptide is generally fused N-terminal to the polypeptideto be targeted (e.g., the fusion partner). However, additional aminoacid residues may be N-terminal to the plastid transit peptide providingthat the fusion protein is at least partially targeted to a plastid. Ina specific embodiment, the plastid transit peptide is in the N-terminalhalf, N-terminal third, or N-terminal quarter of the fusion protein.Most or all of the plastid transit peptide is generally cleaved from thefusion protein upon insertion into the plastid. The position of cleavagemay vary slightly between plant species, at different plantdevelopmental stages, as a result of specific intercellular conditionsor the particular combination of transit peptide/fusion partner used. Inone embodiment, the plastid transit peptide cleavage is homogenous suchthat the cleavage site is identical in a population of fusion proteins.In another embodiment, the plastid transit peptide is not homogenous,such that the cleavage site varies by 1-10 amino acids in a populationof fusion proteins. The plastid transit peptide can be recombinantlyfused to a second protein in one of several ways. For example, arestriction endonuclease recognition site can be introduced into thenucleotide sequence of the transit peptide at a position correspondingto its C-terminal end and the same or a compatible site can beengineered into the nucleotide sequence of the protein to be targeted atits N-terminal end. Care must be taken in designing these sites toensure that the coding sequences of the transit peptide and the secondprotein are kept “in frame” to allow the synthesis of the desired fusionprotein. In some cases, it may be preferable to remove the initiatormethionine codon of the second protein when the new restriction site isintroduced. The introduction of restriction endonuclease recognitionsites on both parent molecules and their subsequent joining throughrecombinant DNA techniques may result in the addition of one or moreextra amino acids between the transit peptide and the second protein.This generally does not affect targeting activity as long as the transitpeptide cleavage site remains accessible and the function of the secondprotein is not altered by the addition of these extra amino acids at itsN-terminus. Alternatively, one skilled in the art can create a precisecleavage site between the transit peptide and the second protein (withor without its initiator methionine) using gene synthesis (Stemmer, etal., (1995) Gene 164:49-53) or similar methods. In addition, the transitpeptide fusion can intentionally include amino acids downstream of thecleavage site. The amino acids at the N-terminus of the mature proteincan affect the ability of the transit peptide to target proteins toplastids and/or the efficiency of cleavage following protein import.This may be dependent on the protein to be targeted. See, e.g., Comai,et al., (1988) J. Biol. Chem. 263(29):15104-9.

In some embodiments, fusion proteins are provided comprising aninsecticidal polypeptide as taught herein, and another insecticidalpolypeptide joined by an amino acid linker. In some embodiments, fusionproteins are provided represented by a formula selected from the groupconsisting of: R¹-L-R², R¹—R² or R²—R¹, wherein R¹ is a polypeptideselected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ IDNO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34,SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO:44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ IDNO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, and SEQ ID NO:72, and R² is another insecticidal polypeptide. The R¹ polypeptide isfused either directly or through a linker (L) segment to the R²polypeptide. The term “directly” defines fusions in which thepolypeptides are joined without a peptide linker. Thus “L” represents achemical bound or polypeptide segment to which both R¹ and R² are fusedin frame, most commonly L is a linear peptide to which R¹ and R² arebound by amide bonds linking the carboxy terminus of R¹ to the aminoterminus of L and carboxy terminus of L to the amino terminus of R². By“fused in frame” is meant that there is no translation termination ordisruption between the reading frames of R¹ and R². The linking group(L) is generally a polypeptide of between 1 and 500 amino acids inlength. The linkers joining the two molecules are preferably designedto: (1) allow the two molecules to fold and act independently of eachother, (2) not have a propensity for developing an ordered secondarystructure which could interfere with the functional domains of the twoproteins, (3) have minimal hydrophobic or charged characteristic whichcould interact with the functional protein domains, and (4) providesteric separation of R¹ and R² such that R¹ and R² could interactsimultaneously with their corresponding receptors on a single cell.Typically surface amino acids in flexible protein regions include Gly,Asn and Ser. Virtually any permutation of amino acid sequencescontaining Gly, Asn and Ser would be expected to satisfy the abovecriteria for a linker sequence. Other neutral amino acids, such as Thrand Ala, may also be used in the linker sequence. Additional amino acidsmay also be included in the linkers due to the addition of uniquerestriction sites in the linker sequence to facilitate construction ofthe fusions.

In some embodiments, the linkers comprise sequences selected from thegroup of formulas: (Gly3Ser)_(n), (Gly4Ser)_(n), (Gly5Ser)_(n),(Gly_(n)Ser)_(n) or (AlaGlySer)_(n) where n is an integer. One exampleof a highly-flexible linker is the (GlySer)-rich spacer region presentwithin the pill protein of the filamentous bacteriophages, e.g.bacteriophages M13 or fd (Schaller, et al., 1975). This region providesa long, flexible spacer region between two domains of the pill surfaceprotein. Also included are linkers in which an endopeptidase recognitionsequence is included. Such a cleavage site may be valuable to separatethe individual components of the fusion to determine if they areproperly folded and active in vitro. Examples of various endopeptidasesinclude, but are not limited to: Plasmin, Enterokinase, Kallikerin,Urokinase, Tissue Plasminogen activator, clostripain, Chymosin,Collagenase, Russell's Viper Venom Protease, Postproline cleavageenzyme, VS protease, Thrombin and factor Xa. In other embodiments,peptide linker segments from the hinge region of heavy chainimmunoglobulins IgG, IgA, IgM, IgD or IgE provide an angularrelationship between the attached polypeptides. The fusion proteins arenot limited by the form, size or number of linker sequences employed andthe only requirement of the linker is that functionally it does notinterfere adversely with the folding and function of the individualmolecules of the fusion.

In another aspect, chimeric proteins are provided that are createdthrough joining two or more portions of the taught insecticidal proteingenes, which originally encoded separate insecticidal proteins to createa chimeric gene. The translation of the chimeric gene results in asingle chimeric protein with regions, motifs, or domains derived fromeach of the original proteins. In certain embodiments, the chimericprotein comprises portions, motifs, or domains of SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ IDNO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32,SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO:42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ IDNO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70,or SEQ ID NO: 72, in any combination.

It is recognized that DNA sequences may be altered by various methods,and that these alterations may result in DNA sequences encoding proteinswith amino acid sequences different than that encoded by the wild-type(or native) pesticidal protein. In some embodiments, an insecticidalprotein taught herein may be altered in various ways including aminoacid substitutions, deletions, truncations, and insertions of one ormore amino acids, including up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, or more amino acid substitutions, deletions and/or insertions orcombinations thereof compared to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ IDNO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44,SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO:54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ IDNO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, and SEQ ID NO: 72.

Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants of a polypeptide can be preparedby mutations in the DNA. This may also be accomplished by one of severalforms of mutagenesis and/or in directed evolution. In some aspects, thechanges encoded in the amino acid sequence will not substantially affectthe function of the protein. Such variants will possess the desiredpesticidal activity. However, it is understood that the ability of ataught polypeptide to confer pesticidal activity may be improved by theuse of such techniques upon the compositions of this disclosure.

For example, conservative amino acid substitutions may be made at one ormore, predicted, nonessential amino acid residues. A “nonessential”amino acid residue is a residue that can be altered from the wild-typesequence of a taught polypeptide without altering the biologicalactivity. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include: amino acidswith basic side chains (e.g., lysine, arginine, histidine); acidic sidechains (e.g., aspartic acid, glutamic acid); polar, negatively chargedresidues and their amides (e.g., aspartic acid, asparagine, glutamic,acid, glutamine; uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine); small aliphatic,nonpolar or slightly polar residues (e.g., Alanine, serine, threonine,praline, glycine); nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, pro line, phenylalanine, methionine, tryptophan); largealiphatic, nonpolar residues (e.g., methionine, leucine, isoleucine,valine, cysteine); beta-branched side chains (e.g., threonine, valine,isoleucine); aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan, histidine); large aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan).

Amino acid substitutions may be made in non-conserved regions thatretain function. In general, such substitutions would not be made forconserved amino acid residues or for amino acid residues residing withina conserved motif, where such residues are essential for proteinactivity. Examples of residues that are conserved and that may beessential for protein activity include, for example, residues that areidentical between all proteins contained in an alignment of similar orrelated toxins to the sequences of the embodiments (e.g., residues thatare identical in an alignment of homologs). Examples of residues thatare conserved but that may allow conservative amino acid substitutionsand still retain activity include, for example, residues that have onlyconservative substitutions between all proteins contained in analignment of similar or related toxins to the sequences of theembodiments (e.g., residues that have only conservative substitutionsbetween all proteins contained in the alignment of the homologs).However, one of skill in the art would understand that functionalvariants may have minor conserved or non-conserved alterations in theconserved residues. Guidance as to appropriate amino acid substitutionsthat do not affect biological activity of the protein of interest may befound in the model of Dayhoff, et al., (1978) Atlas of Protein Sequenceand Structure (Natl. Biomed. Res. Found., Washington, D.C.), hereinincorporated by reference.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, (1982) J Mol Biol.157(1):105-132). It is accepted that the relative hydropathic characterof the amino acid contributes to the secondary structure of theresultant protein, which in turn defines the interaction of the proteinwith other molecules, for example, enzymes, substrates, receptors, DNA,antibodies, antigens, and the like.

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein. Each amino acid has beenassigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte and Doolittle, ibid). These are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); praline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9) and arginine(−4.5). In making such changes, the substitution of amino acids whosehydropathic indices are within +2 is preferred, those which are within+1 are particularly preferred, and those within +0.5 are even moreparticularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, states that the greatest local average hydrophilicity ofa protein, as governed by the hydrophilicity of its adjacent aminoacids, correlates with a biological property of the protein. As detailedin U.S. Pat. No. 4,554,101, the following hydrophilicity values havebeen assigned to amino acid residues: arginine (+3.0); lysine (+3.0);aspartate (+3.0.+0.1); glutamate (+3.0.+0.1); serine (+0.3); asparagine(+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); praline(−0.5.+0.1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); and tryptophan (−3.4).

Alternatively, alterations may be made to the protein sequence of manyproteins at the amino or carboxy terminus without substantiallyaffecting activity. This can include insertions, deletions oralterations introduced by modern molecular methods, such as PCR,including PCR amplifications that alter or extend the protein codingsequence by virtue of inclusion of amino acid encoding sequences in theoligonucleotides utilized in the PCR amplification. Alternatively, theprotein sequences added can include entire protein coding sequences,such as those used commonly in the art to generate protein fusions. Suchfusion proteins are often used to (1) increase expression of a proteinof interest (2) introduce a binding domain, enzymatic activity orepitope to facilitate either protein purification, protein detection orother experimental uses known in the art (3) target secretion ortranslation of a protein to a subcellular organelle, such as theperiplasmic space of Gram-negative bacteria, mitochondria orchloroplasts of plants or the endoplasmic reticulum of eukaryotic cells,the latter of which often results in glycosylation of the protein.

Variant nucleotide and amino acid sequences of the disclosure alsoencompass sequences derived from mutagenic and recombinogenic proceduressuch as DNA shuffling. With such a procedure, one or more differentinsecticidal polypeptide coding regions can be used to create a newpolypeptide possessing the desired properties. In this manner, librariesof recombinant polynucleotides are generated from a population ofrelated sequence polynucleotides comprising sequence regions that havesubstantial sequence identity and can be homologously recombined invitro or in vivo. For example, using this approach, sequence motifsencoding a domain of interest may be shuffled between a pesticidal geneand other known pesticidal genes to obtain a new gene coding for aprotein with an improved property of interest, such as an increasedinsecticidal activity. Strategies for such DNA shuffling are known inthe art. See, for example, Stemmer, (1994) Proc. Natl. Acad. Sci. USA91:10747-10751; Stemmer, (1994) Nature 370:389-391; Crameri, et al.,(1997) Nature Biotech. 15:436-438; Moore, et al., (1997) J. Mol. Biol.272:336-347; Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA94:4504-4509; Crameri, et al., (1998) Nature 391:288-291; and U.S. Pat.Nos. 5,605,793 and 5,837,458.

Domain swapping or shuffling is another mechanism for generating alteredpolypeptides. Domains may be swapped between polypeptides, resulting inhybrid or chimeric toxins with improved insecticidal activity or targetspectrum. Methods for generating recombinant proteins and testing themfor pesticidal activity are well known in the art (see, for example,Naimov, et al., (2001) Appl. Environ. Microbiol. 67:5328-5330; de Maagd,et al., (1996) Appl. Environ. Microbiol. 62:1537-1543; Ge, et al.,(1991) J. Biol. Chem. 266:17954-17958; Schnepf, et al., (1990) J. Biol.Chem. 265:20923-20930; Rang, et al., 91999) Appl. Environ. Microbiol.65:2918-2925).

Both DNA shuffling and site-directed mutagenesis can be used to definepolypeptide sequences that possess pesticidal activity. The personskilled in the art will be able to use comparisons to other proteins orfunctional assays to further define motifs. High throughput screeningcan be used to test variations of those motifs to determine the role ofspecific residues.

Receptor Identification and Isolation

Receptors to the taught insecticidal proteins, or to variants orfragments thereof, are also encompassed. Methods for identifyingreceptors are well known in the art (see, Hofmann, et. al., (1988) Eur.J. Biochem. 173:85-91; Gill, et al., (1995) J. Biol. Chem. 27277-27282)and can be employed to identify and isolate the receptor that recognizesthe taught insecticidal proteins using the brush-border membranevesicles from susceptible insects. In addition to the radioactivelabeling method listed in the cited literature, taught proteins can belabeled with fluorescent dye and other common labels such asstreptavidin. Brush-border membrane vesicles (BBMV) of susceptibleinsects such as soybean looper and stink bugs can be prepared accordingto the protocols listed in the references and separated on SDS-PAGE geland blotted on suitable membrane. Labeled proteins can be incubated withblotted membrane of BBMV and identified with the labeled reporters.Identification of protein band(s) that interact with the proteins can bedetected by N-terminal amino acid gas phase sequencing or massspectrometry based protein identification method (Patterson, (1998)10.22, 1-24, Current Protocol in Molecular Biology published by JohnWiley & Son Inc). Once the protein is identified, the corresponding genecan be cloned from genomic DNA or cDNA library of the susceptibleinsects and binding affinity can be measured directly with the proteins.Receptor function for insecticidal activity by the taught proteins canbe verified by an RNAi type of gene knock out method (Rajagopal, et al.,(2002) J. Biol. Chem. 277:46849-46851).

Nucleotide Constructs, Expression Cassettes, and Vectors

The use of the term “nucleotide constructs” herein is not intended tolimit the embodiments to nucleotide constructs comprising DNA. Those ofordinary skill in the art will recognize that nucleotide constructsparticularly polynucleotides and oligonucleotides composed ofribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides may also be employed in the methods disclosedherein. The nucleotide constructs, nucleic acids, and nucleotidesequences of the embodiments additionally encompass all complementaryforms of such constructs, molecules, and sequences. Further, thenucleotide constructs, nucleotide molecules, and nucleotide sequences ofthe embodiments encompass all nucleotide constructs, molecules, andsequences which can be employed in the methods of the embodiments fortransforming plants including, but not limited to, those comprised ofdeoxyribonucleotides, ribonucleotides, and combinations thereof. Suchdeoxyribonucleotides and ribonucleotides include both naturallyoccurring molecules and synthetic analogues. The nucleotide constructs,nucleic acids, and nucleotide sequences of the embodiments alsoencompass all forms of nucleotide constructs including, but not limitedto, single-stranded forms, double-stranded forms, hairpins,stem-and-loop structures and the like.

A further embodiment relates to a transformed organism such as anorganism selected from plant and insect cells, bacteria, yeast,baculovirus, protozoa, nematodes and algae. The transformed organismcomprises a DNA molecule of the embodiments, an expression cassettecomprising the DNA molecule or a vector comprising the expressioncassette, which may be stably incorporated into the genome of thetransformed organism.

The sequences of the embodiments are provided in DNA constructs forexpression in the organism of interest. The construct will include 5′and 3′ regulatory sequences operably linked to a sequence of theembodiments. The term “operably linked” as used herein refers to afunctional linkage between a promoter and a second sequence, wherein thepromoter sequence initiates and mediates transcription of the DNAsequence corresponding to the second sequence. Generally, operablylinked means that the nucleic acid sequences being linked are contiguousand were necessary to join two protein-coding regions in the samereading frame. The construct may additionally contain at least oneadditional gene to be co-transformed into the organism. Alternatively,the additional gene(s) can be provided on multiple DNA constructs.

In some embodiments, the DNA construct comprises a polynucleotideencoding an insecticidal protein taught herein, which is operably linkedto a heterologous regulatory sequence.

In some embodiments, the DNA construct comprises a polynucleotideencoding an insecticidal protein taught herein, which is operably linkedto a heterologous regulatory sequence, said polynucleotide selected fromthe group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ IDNO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35,SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO:45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ IDNO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, and SEQ ID NO: 71, or asequence corresponding to the aforementioned that has been codonoptimized for expression in a host cell of interest, for example a plantcell in some embodiments.

In some embodiments, the DNA construct comprises a polynucleotideencoding an insecticidal protein taught herein, which is operably linkedto a heterologous regulatory sequence, said protein selected from thegroup consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ IDNO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36,SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO:46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ IDNO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, and SEQ ID NO: 72, or a variantthereof.

Such a DNA construct is provided with a plurality of restriction sitesfor insertion of the polypeptide gene sequence to be under thetranscriptional regulation of the regulatory regions. The DNA constructmay additionally contain selectable marker genes.

The DNA construct will generally include in the 5′ to 3′ direction oftranscription: a transcriptional and translational initiation region(i.e., a promoter), a DNA sequence of the embodiments, and atranscriptional and translational termination region (i.e., terminationregion) functional in the organism serving as a host, e.g. a bacterialcell or plant cell.

The transcriptional initiation region (i.e., the promoter) may benative, analogous, foreign, or heterologous to the host organism and/orto the sequence of the embodiments. Additionally, the promoter may bethe natural sequence or alternatively a synthetic sequence. The term“foreign” as used herein indicates that the promoter is not found in thenative organism into which the promoter is introduced. Where thepromoter is “heterologous” to the sequence of the embodiments, it isintended that the promoter is not the native or naturally occurringpromoter for the operably linked sequence of the embodiments (i.e., notthe native location). As used herein, a chimeric gene comprises a codingsequence operably linked to a transcription initiation region that isheterologous to the coding sequence. Where the promoter is a native ornatural sequence, the expression of the operably linked sequence isaltered from the wild-type expression, which results in an alteration inphenotype.

In some embodiments, the DNA construct may also include atranscriptional enhancer sequence. As used herein, the term an“enhancer” refers to a DNA sequence which can stimulate promoteractivity, and may be an innate element of the promoter or a heterologouselement inserted to enhance the level or tissue-specificity of apromoter. Various enhancers are known in the art including for example,intrans with gene expression enhancing properties in plants (US PatentApplication Publication Number 2009/0144863, the ubiquitin intron (i.e.,the maize ubiquitin intron 1 (see, for example, NCBI sequence 594464;Christensen and Quail (1996) Transgenic Res. 5:213-218; Christensen etal. (1992) Plant Molecular Biology 18:675-689)), the omega enhancer orthe omega prime enhancer (Gallie, et al., (1989) Molecular Biology ofRNA ed. Cech (Liss, New York) 237-256 and Gallie, et al., (1987) Gene60:217-25), the CaMV 35S enhancer (see, e.g., Benfey, et al., (1990)EMBO J. 9: 1685-96), the maize Adhl intron (Kyozuka et al. (1991) Mol.Gen. Genet. 228:40-48; Kyozuka et al. (1990) Maydica 35:353-357), theenhancers of U.S. Pat. No. 7,803,992, and the sugarcane bacilliformviral (SCBV) enhancer of WO2013130813 may also be used, each of which isincorporated by reference. The above list of transcriptional enhancersis not meant to be limiting. Any appropriate transcriptional enhancercan be used in the embodiments.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,may be native with the plant host, or may be derived from another source(i.e., foreign or heterologous to the promoter, the sequence ofinterest, the plant host or any combination thereof).

Convenient termination regions are available from the Ti-plasmid of A.tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See also, Guerineau, et al., (1991) Mol. Gen.Genet. 262:141-144; Proudfoot, (1991) Cell 64:671-674; Sanfacon, et al.,(1991) Genes Dev. 5:141-149; Magen, et al., (1990) Plant Cell2:1261-1272; Munroe, et al., (1990) Gene 91:151-158; Ballas, et al.,(1989) Nucleic Acids Res. 17:7891-7903 and Joshi, et al., (1987) NucleicAcid Res. 15:9627-9639.

Where appropriate, a nucleic acid may be optimized for increasedexpression in the host organism. Thus, where the host organism is aplant, the synthetic nucleic acids can be synthesized usingplant-preferred codons for improved expression. See, for example,Campbell and Gowri, (1990) Plant Physiol. 92:1-11 for a discussion ofhost preferred codon usage. For example, although nucleic acid sequencesof the embodiments may be expressed in both monocotyledonous anddicotyledonous plant species, sequences can be modified to account forthe specific codon preferences and GC content preferences ofmonocotyledons or dicotyledons, as these preferences have been shown todiffer (Murray et al. (1989) Nucleic Acids Res. 17:477-498).

Thus, one of skill in the art will understand how to utilize specificplant codon usage tables to derive the optimal sequences to express theinsecticidal proteins of the disclosure. See, e.g. US 2016/0366891 (U.S.application Ser. No. 15/022,109), which is hereby incorporated byreference in its entirety.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon like repeats, and other well characterized sequences that maybe deleterious to gene expression. The GC content of the sequence may beadjusted to levels average for a given cellular host, as calculated byreference to known genes expressed in the host cell. The term “hostcell” as used herein refers to a cell which contains a vector andsupports the replication and/or expression of the expression vector.Host cells may be prokaryotic cells such as E. coli or eukaryotic cellssuch as yeast, insect, amphibian or mammalian cells or monocotyledonousor dicotyledonous plant cells. An example of a monocotyledonous hostcell is a maize host cell. When possible, the sequence is modified toavoid predicted hairpin secondary mRNA structures.

The expression cassettes may additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include: picomavirus leaders, forexample, EMCV leader (Encephalomyocarditis 5′ noncoding region)(Elroy-Stein, et al., (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130);potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie,et al., (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf MosaicVirus), human immunoglobulin heavy-chain binding protein (BiP) (Macejak,et al., (1991) Nature 353:90-94); untranslated leader from the coatprotein mRNA of alfalfa mosaic virus (AMY RNA 4) (Jobling, et al.,(1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie,et al., (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York),pp. 237-256) and maize chlorotic mottle virus leader (MCMV) (Lommel, etal., (1991) Virology 81:382-385). See also, Della-Cioppa, et al., (1987)Plant Physiol. 84:965-968.

Such constructs may also contain a “signal sequence” or “leadersequence” to facilitate co-translational or post-translational transportof the peptide to certain intracellular structures such as thechloroplast (or other plastid), endoplasmic reticulum or Golgiapparatus. “Signal sequence” as used herein refers to a sequence that isknown or suspected to result in cotranslational or post-translationalpeptide transport across the cell membrane. In eukaryotes, thistypically involves secretion into the Golgi apparatus, with someresulting glycosylation. Insecticidal toxins of bacteria are oftensynthesized as protoxins, which are protolytically activated in the gutof the target pest (Chang, (1987) Methods Enzymol. 153:507-516). In someembodiments, the signal sequence is located in the native sequence ormay be derived from a sequence of the embodiments. “Leader sequence” asused herein refers to any sequence that when translated, results in anamino acid sequence sufficient to trigger co-translational transport ofthe peptide chain to a subcellular organelle. Thus, this includes leadersequences targeting transport and/or glycosylation by passage into theendoplasmic reticulum, passage to vacuoles, plastids includingchloroplasts, mitochondria, and the like. Nuclear encoded proteinstargeted to the chloroplast thylakoid lumen compartment have acharacteristic bipartite transit peptide, composed of a stromaltargeting signal peptide and a lumen targeting signal peptide. Thestromal targeting information is in the amino-proximal portion of thetransit peptide. The lumen targeting signal peptide is in thecarboxyl-proximal portion of the transit peptide, and contains all theinformation for targeting to the lumen. Recent research in proteomics ofthe higher plant chloroplast has achieved the identification of numerousnuclear encoded lumen proteins (Kieselbach et al. FEBS Lett.480:271-276, 2000; Peltier et al. Plant Cell 12:319-341, 2000; Brickeret al. Biochim. Biophys. Acta 1503:350-356, 2001), the lumen targetingsignal peptide of which can potentially be used in accordance with thepresent disclosure. About 80 proteins from Arabidopsis, as well ashomologous proteins from spinach and garden pea, are reported byKieselbach et al., Photosynthesis Research, 78:249-264, 2003. Inparticular, Table 2 of this publication, which is incorporated into thedescription herewith by reference, discloses 85 proteins from thechloroplast lumen, identified by their accession number (see also USPatent Application Publication 2009/09044298). In addition, thepublished draft version of the rice genome (Goff et al, Science296:92-100, 2002) is a suitable source for lumen targeting signalpeptide which may be used in accordance with the present disclosure.

Suitable chloroplast transit peptides (CTP) are well known to oneskilled in the art also include chimeric CTPs comprising but not limitedto, an N-terminal domain, a central domain or a C-terminal domain from aCTP from Oryza sativa-l-deoxy-D xyulose-5-Phosphate Synthase, Oryzasativa-Superoxide dismutase, Oryza sativa-soluble starch synthase, Oryzasativa-NADP-dependent Malic acid enzyme, Oryzasativa-Phospho-2-dehydro-3-deoxyheptonate Aldolase 2, Oryzasativa-L-Ascorbate peroxidase 5, Oryza sativa-Phosphoglucan waterdikinase, Zea Mays ssRUBISCO, Zea Mays-beta-glucosidase, Zea Mays-Malatedehydrogenase, Zea Mays Thioredoxin M-type (US Patent ApplicationPublication 2012/0304336). Chloroplast transit peptides of US PatentPublications US20130205440A1, US20130205441A1 and US20130210114A1. Thepolypeptide gene to be targeted to the chloroplast may be optimized forexpression in the chloroplast to account for differences in codon usagebetween the plant nucleus and this organelle.

In preparing the expression cassette, the various DNA fragments may bemanipulated so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

Promoters

A number of promoters can be used in the practice of the embodiments.The promoters can be selected based on the desired outcome. The nucleicacids can be combined with constitutive, tissue-preferred, inducible, orother promoters for expression in the host organism. Promoters of thepresent invention include homologues of cis elements known to effectgene regulation that show homology with the promoter sequences of thepresent invention. These cis elements include, but are not limited to,oxygen responsive cis elements (Cowen et al., J. Biol. Chem.268(36):26904-26910 (1993)), light regulatory elements (Bruce andQuaill, Plant Cell 2 (11):1081-1089 (1990); Bruce et al., EMBO J.10:3015-3024 (1991); Rocholl et al., Plant Sci. 97:189-198 (1994); Blocket al., Proc. Natl. Acad. Sci. USA 87:5387-5391 (1990); Giuliano et al.,Proc. Natl. Acad. Sci. USA 85:7089-7093 (1988); Staiger et al., Proc.Natl. Acad. Sci. USA 86:6930-6934 (1989); Izawa et al., Plant Cell6:1277-1287 (1994); Menkens et al., Trends in Biochemistry 20:506-510(1995); Foster et al., FASEB J. 8:192-200 (1994); Plesse et al., Mol.Gen. Gene. 254:258-266 (1997); Green et al., EMBO J. 6:2543-2549 (1987);Kuhlemeier et al., Ann. Rev. Plant Physiol. 38:221-257 (1987); Villainet al., J. Biol. Chem. 271:32593-32598 (1996); Lam et al., Plant Cell2:857-866 (1990); Gilmartin et al., Plant Cell 2:369-378 (1990); Dattaet al., Plant Cell 1:1069-1077 (1989); Gilmartin et al., Plant Cell2:369-378 (1990); Castresana et al., EMBO J. 7:1929-1936 (1988); Ueda etal., Plant Cell 1:217-227 (1989); Terzaghi et al., Annu. Rev. PlantPhysiol. Plant Mol. Biol. 46:445-474 (1995); Green et al., EMBO J.6:2543-2549 (1987); Villain et al., J. Biol. Chem. 271:32593-32598(1996); Tjaden et al., Plant Cell 6:107-118 (1994); Tjaden et al., PlantPhysiol. 108:1109-1117 (1995); Ngai et al., Plant J. 12:1021-1234(1997); Bruce et al., EMBO J. 10:3015-3024 (1991); Ngai et al., Plant J.12:1021-1034 (1997)), elements responsive to gibberellin, (Muller etal., J. Plant Physiol. 145:606-613 (1995); Croissant et al., PlantScience 116:27-35 (1996); Lohmer et al., EMBO J. 10:617-624 (1991);Rogers et al., Plant Cell 4:1443-1451 (1992); Lanahan et al., Plant Cell4:203-211 (1992); Skriver et al., Proc. Natl. Acad. Sci. USA88:7266-7270 (1991); Gilmartin et al., Plant Cell 2:369-378 (1990);Huang et al., Plant Mol. Biol. 14:655-668 (1990), Gubler et al., PlantCell 7:1879-1891 (1995)), elements responsive to abscisic acid, (Busk etal., Plant Cell 9:2261-2270 (1997); Guiltinan et al., Science250:267-270 (1990); Shen et al., Plant Cell 7:295-307 (1995); Shen etal., Plant Cell 8:1107-1119 (1996); Seo et al., Plant Mol. Biol.27:1119-1131 (1995); Marcotte et al., Plant Cell 1:969-976 (1989); Shenet al., Plant Cell 7:295-307 (1995); Iwasaki et al., Mol Gen Genet247:391-398 (1995); Hattori et al., Genes Dev. 6:609-618 (1992); Thomaset al., Plant Cell 5:1401-1410 (1993)), elements similar to abscisicacid responsive elements, (Ellerstrom et al., Plant Mol. Biol.32:1019-1027 (1996)), auxin responsive elements (Liu et al., Plant Cell6:645-657 (1994); Liu et al., Plant Physiol. 115:397-407 (1997); Kosugiet al., Plant J. 7:877-886 (1995); Kosugi et al., Plant Cell 9:1607-1619(1997); Ballas et al., J. Mol. Biol. 233: 580-596 (1993)), a cis elementresponsive to methyl jasmonate treatment (Beaudoin and Rothstein, PlantMol. Biol. 33:835-846 (1997)), a cis element responsive to abscisic acidand stress response (Straub et al., Plant Mol. Biol. 26:617-630 (1994)),ethylene responsive cis elements (Itzhaki et al., Proc. Natl. Acad. Sci.USA 91:8925-8929 (1994); Montgomery et al., Proc. Natl. Acad. Sci. USA90:5939-5943 (1993); Sessa et al., Plant Mol. Biol. 28:145-153 (1995);Shinshi et al., Plant Mol. Biol. 27:923-932 (1995)), salicylic acid cisresponsive elements, (Strange et al., Plant J. 11:1315-1324 (1997); Qinet al., Plant Cell 6:863-874 (1994)), a cis element that responds towater stress and abscisic acid (Lam et al., J. Biol. Chem. 266:17131-17135 (1991); Thomas et al., Plant Cell 5:1401-1410 (1993); Pia etal., Plant Mol Biol. 21:259-266 (1993)), a cis element essential for Mphase-specific expression (Ito et al., Plant Cell 10:331-341 (1998)),sucrose responsive elements (Huang et al., Plant Mol. Biol. 14:655-668(1990); Hwang et al., Plant Mol Biol 36:331-341 (1998); Grierson et al.,Plant J. 5:815-826 (1994)), heat shock response elements (Pelham et al.,Trends Genet. 1:31-35 (1985)), elements responsive to auxin and/orsalicylic acid and also reported for light regulation (Lam et al., Proc.Natl. Acad. Sci. USA 86:7890-7897 (1989); Benfey et al., Science250:959-966 (1990)), elements responsive to ethylene and salicylic acid(Ohme-Takagi et al., Plant Mol. Biol. 15:941-946 (1990)), elementsresponsive to wounding and abiotic stress (Laake et al., Proc. Natl.Acad. Sci. USA 89:9230-9234 (1992); Mhiri et al., Plant Mol. Biol.33:257-266 (1997)), antioxidant response elements (Rushmore et al., J.Biol. Chem. 266: 11632-11639; Dalton et al., Nucleic Acids Res.22:5016-5023 (1994)), Sph elements (Suznki et al., Plant Cell 9:799-8071997)), elicitor responsive elements, (Fnkuda et al., Plant Mol. Biol.34:81-87 (1997); Rushton et al., EMBO J. 15:5690-5700 (1996)), metalresponsive elements (Stuart et al., Nature 317:828-831 (1985); Westin etal., EMBO J. 7:3763-3770 (1988); Thiele et al., Nucleic Acids Res.20:1183-1191 (1992); Faisst et al., Nucleic Acids Res. 20:3-26 (1992)),low temperature responsive elements, (Baker et al., Plant Mol. Biol.24:701-713 (1994); Jiang et al., Plant Mol. Biol. 30:679-684 (1996);Nordin et al., Plant Mol. Biol. 21:641-653 (1993); Zhou et al., J. Biol.Chem. 267:23515-23519 (1992)), drought responsive elements, (Yamaguchiet al., Plant Cell 6:251-264 (1994); Wang et al., Plant Mol. Biol.28:605-617 (1995); Bray E A, Trends in Plant Science 2:48-54 (1997))enhancer elements for glutenin, (Colot et al., EMBO J. 6:3559-3564(1987); Thomas et al., Plant Cell 2:1171-1180 (1990); Kreis et al.,Philos. Trans. R. Soc. Lond., B314:355-365 (1986)), light-independentregulatory elements, (Lagrange et al., Plant Cell 9:1469-1479 (1997);Villain et al., J. Biol. Chem. 271: 32593-32598 (1996)), OCS enhancerelements, (Bouchez et al., EMBO J. 8:4197-4204 (1989); Foley et al.,Plant J. 3:669-679 (1993)), ACGT elements, (Foster et al., FASEB J.8:192-200 (1994); Izawa et al., Plant Cell 6:1277-1287 (1994); Izawa etal., J. Mol. Biol. 230:1131-1144 (1993)), negative cis elements inplastid related genes, (Zhou et al., J. Biol. Chem. 267:23515-23519(1992); Lagrange et al., Mol. Cell Biol. 13:2614-2622 (1993); Lagrangeet al., Plant Cell 9:1469-1479 (1997); Zhou et al., J. Biol. Chem. 267:23515-23519 (1992)), prolamin box elements, (Forde et al., Nucleic AcidsRes. 13:7327-7339 (1985); Colot et al., EMBO J. 6:3559-3564 (1987);Thomas et al., Plant Cell 2:1171-1180 (1990); Thompson et al., PlantMol. Biol. 15:755-764 (1990); Vicente et al., Proc. Natl. Acad. Sci. USA94:7685-7690 (1997)), elements in enhancers from the IgM heavy chaingene (Gillies et al., Cell 33:717-728 (1983); Whittier et al., NucleicAcids Res. 15:2515-2535 (1987)).

Examples of promoters include those described in: U.S. Pat. No.6,437,217 (maize RS81 promoter), U.S. Pat. No. 5,641,876 (rice actinpromoter), U.S. Pat. No. 6,426,446 (maize RS324 promoter), U.S. Pat. No.6,429,362 (maize PR-1 promoter), U.S. Pat. No. 6,232,526 (maize A3promoter), U.S. Pat. No. 6,177,611 (constitutive maize promoters), U.S.Pat. Nos. 5,322,938, 5,352,605, 5,359,142 and 5,530,196 (35S promoter),U.S. Pat. No. 6,433,252 (maize L3 oleosin promoter, P-Zm.L3), U.S. Pat.No. 6,429,357 (rice actin 2 promoter as well as a rice actin 2 intron),U.S. Pat. No. 5,837,848 (root specific promoter), U.S. Pat. No.6,294,714 (light inducible promoters), U.S. Pat. No. 6,140,078 (saltinducible promoters), U.S. Pat. No. 6,252,138 (pathogen induciblepromoters), U.S. Pat. No. 6,175,060 (phosphorus deficiency induciblepromoters), U.S. Pat. No. 6,635,806 (gama-coixin promoter, P-CI.Gcx),U.S. patent application Ser. No. 09/757,089 (maize chloroplast aldolasepromoter), and U.S. Pat. No. 8,772,466 (maize transcription factorNuclear Factor B (NFB2)).

Suitable constitutive promoters for use in a plant host cell include,for example, the core promoter of the Rsyn7 promoter and otherconstitutive promoters disclosed in WO 1999/43838 and U.S. Pat. No.6,072,050; the core CaMV 35S promoter (Odell, et al., (1985) Nature313:810-812); rice actin (McElroy, et al., (1990) Plant Cell 2:163-171);ubiquitin (Christensen, et al., (1989) Plant Mol. Biol. 12:619-632 andChristensen, et al., (1992) Plant Mol. Biol. 18:675-689); pEMU (Last, etal., (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten, et al., (1984)EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026) and thelike.

Other constitutive promoters include, for example, those discussed in:U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604, 121; 5,569,597; 5,466,785;5,399,680; 5,268,463; 5,608,142 and 6,177,611.

Suitable constitutive promoters also include promoters that have strongexpression in nearly all tissues but have low expression in pollen,including but not limited to: Banana Streak Virus (Acuminata Yunnan)promoters (BSV(AY)) disclosed in US patent U.S. Pat. No. 8,338,662;Banana Streak Virus (Acuminata Vietnam) promoters (BSV (AV)) disclosedin US patent U.S. Pat. No. 8,350,121; and Banana Streak Virus (Mysore)promoters (BSV(M YS)) disclosed in US patent U.S. Pat. No. 8,395,022.

Depending on the desired outcome, it may be beneficial to express thegene from an inducible promoter. Of particular interest for regulatingthe expression of the nucleotide sequences of the embodiments in plantsare wound inducible promoters. Such wound inducible promoters, mayrespond to damage caused by insect feeding, and include potatoproteinase inhibitor (pin II) gene (Ryan, (1990) Ann. Rev. Phytopath.28:425-449; Duan, et al., (1996) Nature Biotechnology 14:494-498); wun1and wun2, U.S. Pat. No. 5,428,148; win1 and win2 (Stanford, et al.,(1989) Mol. Gen. Genet. 215:200-208); systemin (McGurl, et al., (1992)Science 225:1570-1573); WIP1 (Rohmeier, et al., (1993) Plant Mol. Biol.22:783-792; Eckelkamp, et al., (1993) FEBS Letters 323:73-76); MPI gene(Corderok, et al., (1994) Plant J. 6(2):141-150) and the like, hereinincorporated by reference.

Additionally, pathogen inducible promoters may be employed in themethods and nucleotide constructs of the embodiments. Such pathogeninducible promoters include those from pathogenesis related proteins (PRproteins), which are induced following infection by a pathogen; e.g., PRproteins, SAR proteins, beta-1,3-glucanase, chitinase, etc. See, forexample, Redolfi, et al., (1983) Neth. J. Plant Pathol. 89:245-254;Uknes, et al., (1992) Plant Cell 4: 645-656 and Van Loon, (1985) PlantMol. Biol. 4:111-116. See also, WO 1999/43819, herein incorporated byreference.

Of interest are promoters that are expressed locally at or near the siteof pathogen infection. See, for example, Marineau, et al., (1987) PlantMol. Biol. 9:335-342; Matton, et al., (1989) Molecular Plant-MicrobeInteractions 2:325-331; Somsisch, et al., (1986) Proc. Natl. Acad. Sci.USA 83:2427-2430; Somsisch, et al., (1988) Mol. Gen. Genet. 2:93-98 andYang, (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen,et al., (1996) Plant J. 10:955-966; Zhang, et al., (1994) Proc. Natl.Acad. Sci. USA 91:2507-2511; Warner, et al., (1993) Plant J. 3:191-201;Siebertz, et al., (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386(nematode-inducible) and the references cited therein. Of particularinterest is the inducible promoter for the maize PRms gene, whoseexpression is induced by the pathogen Fusarium moniliforme (see, forexample, Cordero, et al., (1992) Physiol. Mol. Plant Path. 41:189-200).

Chemical regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be a chemicalinducible promoter, where application of the chemical induces geneexpression or a chemical repressible promoter, where application of thechemical represses gene expression. Chemical inducible promoters areknown in the art and include, but are not limited to, the maize In2-2promoter, which is activated by benzenesulfonamide herbicide safeners,the maize GST promoter, which is activated by hydrophobic electrophiliccompounds that are used as pre-emergent herbicides, and the tobaccoPR-la promoter, which is activated by salicylic acid. Other chemicalregulated promoters of interest include steroid responsive promoters(see, for example, the glucocorticoid-inducible promoter in Schena, etal., (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis, etal., (1998) Plant J. 14(2):247-257) and tetracycline-inducible andtetracycline-repressible promoters (see, for example, Gatz, et al.,(1991) Mol. Gen. Genet. 227:229-237 and U.S. Pat. Nos. 5,814,618 and5,789,156), herein incorporated by reference.

Tissue preferred promoters can be utilized to target enhancedpolypeptide expression within a particular plant tissue. Tissuepreferred promoters include those discussed in Yamamoto, et al., (1997)Plant J. 12(2)255-265; Kawamata, et al., (1997) Plant Cell Physiol.38(7):792-803; Hansen, et al., (1997) Mol. Gen Genet. 254(3):337-343;Russell, et al., (1997) Transgenic Res. 6(2):157-168; Rinehart, et al.,(1996) Plant Physiol. 112(3):1331-1341; Van Camp, et al., (1996) PlantPhysiol. 112(2):525-535; Canevascini, et al., (1996) Plant Physiol.112(2):513-524; Yamamoto, et al., (1994) Plant Cell Physiol.35(5):773-778; Lam, (1994) Results Probl. Cell Differ. 20: 181-196;Orozco, et al., (1993) Plant Mol Biol. 23(6): 1129-1138; Matsuoka, etal., (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590 andGuevara-Garcia, et al., (1993) Plant J. 4(3):495-505. Such promoters canbe modified, if necessary, for weak expression if desired.

Leaf preferred promoters are known in the art. See, for example,Yamamoto, et al., (1997) Plant J. 12(2):255-265; Kwon, et al., (1994)Plant Physiol. 105:357-67; Yamamoto, et al., (1994) Plant Cell Physiol.35(5):773-778; Gator, et al., (1993) Plant J. 3:509-18; Orozco, et al.,(1993) Plant Mol. Biol. 23(6):1129-1138 and Matsuoka, et al., (1993)Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

Root preferred or root specific promoters are known and can be selectedfrom the many available from the literature or isolated de novo fromvarious compatible species. See, for example, Hire, et al., (1992) PlantMol. Biol. 20(2):207-218 (soybean root specific glutamine synthetasegene); Keller and Baumgartner, (1991) Plant Cell 3(10):1051-1061 (rootspecific control element in the GRP 1.8 gene of French bean); Sanger, etal., (1990) Plant Mol. Biol. 14(3):433-443 (root specific promoter ofthe mannopine synthase (MAS) gene of Agrobacterium tumefaciens) andMiao, et al., (1991) Plant Cell 3(1):11-22 (full length cDNA cloneencoding cytosolic glutamine synthetase (GS), which is expressed inroots and root nodules of soybean). See also, Bogusz, et al., (1990)Plant Cell 2(7): 633-641, where two root specific promoters isolatedfrom hemoglobin genes from the nitrogen-fixing nonlegume Parasponiaandersonii and the related non-nitrogen-fixing nonlegume Trema tomentosaare described. The promoters of these genes were linked to aβ-glucuronidase reporter gene and introduced into both the nonlegumeNicotiana tabacum and the legume Lotus corniculatus, and in bothinstances root specific promoter activity was preserved. Leach andAoyagi, (1991) describe their analysis of the promoters of the highlyexpressed rolC and rolD root inducing genes of Agrobacterium rhizogenes(see, Plant Science (Limerick) 79(1):69-76). They concluded thatenhancer and tissue-preferred DNA determinants are dissociated in thosepromoters. Teeri, et al., (1989) used gene fusion to lacZ to show thatthe Agrobacterium T-DNA gene encoding octopine synthase is especiallyactive in the epidermis of the root tip and that the TR2′ gene is rootspecific in the intact plant and stimulated by wounding in leaf tissue,an especially desirable combination of characteristics for use with aninsecticidal or larvicidal gene (see, EMBO J. 8(2): 343-350). The TRI′gene fused to nptll (neomycin phosphotransferase II) showed similarcharacteristics. Additional root preferred promoters include theVfENOD-GRP3 gene promoter (Kuster, et al., (1995) Plant Mol. Biol.29(4):759-772) and rolB promoter (Capana, et al., (1994) Plant Mol.Biol. 25(4):681-691. See also, U.S. Pat. Nos. 5,837,876; 5,750,386;5,633,363; 5,459,252; 5,401,836; 5,110,732 and 5,023,179. Arabidopsisthaliana root preferred regulatory sequences are disclosed in US PatentApplication US20130117883. Root preferred sorghum (Sorghum bicolor) RCc3promoters are disclosed in US Patent Application US2012/0210463. Theroot preferred maize promoters of US Patent Application Publication2003/0131377, U.S. Pat. Nos. 7,645,919, and 8,735,655. The root capspecific 1 (ZmRCPl) maize promoters of US Patent Application Publication2013/0025000. The root preferred maize promoters of US PatentApplication Publication 2013/0312136.

“Seed preferred” promoters include both “seed-specific” promoters (thosepromoters active during seed development such as promoters of seedstorage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See, Thompson, et al., (1989)BioEssays 10:108, herein incorporated by reference. Such seed preferredpromoters include, but are not limited to, Cim1 (cytokinin-inducedmessage); cZ19B1 (maize 19 kDa zein); and milps(myo-inositol-1-phosphate synthase) (see, U.S. Pat. No. 6,225,529,herein incorporated by reference). Gamma-zein and Glb-1 areendosperm-specific promoters. For dicots, seed specific promotersinclude, but are not limited to, Kunitz trypsin inhibitor 3 (KTi3)(Jofuku and Goldberg, (1989) Plant Cell 1:1079-1093), bean β-phaseolin,napin, β-conglycinin, glycinin 1, soybean lectin, cruciferin, and thelike. For monocots, seed specific promoters include, but are not limitedto, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken1, shrunken 2, globulin 1, etc. See also, WO 2000/12733, where seedpreferred promoters from end1 and end2 genes are disclosed; hereinincorporated by reference. In dicots, seed specific promoters include,but are not limited to, seed coat promoter from Arabidopsis, pBAN; andthe early seed promoters from Arabidopsis, p26, p63, and p63tr (U.S.Pat. Nos. 7,294,760 and 7,847,153). A promoter that has “preferred”expression in a particular tissue is expressed in that tissue to agreater degree than in at least one other plant tissue. Some tissuepreferred promoters show expression almost exclusively in the particulartissue.

Where low level expression is desired, weak promoters will be used.Generally, the term “weak promoter” as used herein refers to a promoterthat drives expression of a coding sequence at a low level. By low levelexpression at levels of about 1/1000 transcripts to about 1/100,000transcripts to about 1/500,000 transcripts is intended. Alternatively,it is recognized that the term “weak promoters” also encompassespromoters that drive expression in only a few cells and not in others togive a total low level of expression. Where a promoter drives expressionat unacceptably high levels, portions of the promoter sequence can bedeleted or modified to decrease expression levels. Such weakconstitutive promoters include, for example the core promoter of theRsyn7 promoter (WO 1999/43838 and U.S. Pat. No. 6,072,050), the core 35SCaMV promoter, and the like. Other constitutive promoters include, forexample, those disclosed in U.S. Pat. Nos. 5,608,149; 5,608,144;5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142 and6,177,611, herein incorporated by reference.

The above list of promoters is not meant to be limiting. Any appropriatepromoter can be used in the embodiments.

Generally, the expression cassette will comprise a selectable markergene for the selection of transformed cells. Selectable marker genes areutilized for the selection of transformed cells or tissues. Marker genesinclude genes encoding antibiotic resistance, such as those encodingneomycin phosphotransferase II (NEO) and hygromycin phosphotransferase(HPT), as well as genes conferring resistance to herbicidal compounds,such as glufosinate ammonium, bromoxynil, imidazolinones and2,4-dichlorophenoxyacetate (2,4-D). Additional examples of suitableselectable marker genes include, but are not limited to, genes encodingresistance to chloramphenicol (Herrera Estrella, et al., (1983) EMBO J.2:987-992); methotrexate (Herrera Estrella, et al., (1983) Nature303:209-213 and Meijer, et al., (1991) Plant Mol. Biol. 16:807-820);streptomycin (Jones, et al., (1987) Mol. Gen. Genet. 210:86-91);spectinomycin (Bretagne-Sagnard, et al., (1996) Transgenic Res.5:131-137); bleomycin (Hille, et al., (1990) Plant Mol. Biol.7:171-176); sulfonamide (Guerineau, et al., (1990) Plant Mol. Biol.15:127-136); bromoxynil (Stalker, et al., (1988) Science 242:419-423);glyphosate (Shaw, et al., (1986) Science 233:478-481 and U.S. patentapplication Ser. Nos. 10/004,357 and 10/427,692); phosphinothricin(DeBlock, et al., (1987)EMBO J. 6:2513-2518). See generally, Yarranton,(1992) Curr. Opin. Biotech. 3:506-511; Christopherson, et al., (1992)Proc. Natl. Acad. Sci. USA 89:6314-6318; Yao, et al., (1992) Cell71:63-72; Reznikoff, (1992) Mol. Microbiol. 6:2419-2422; Barkley, etal., (1980) in The Operon, pp. 177-220; Hu, et al., (1987) Cell48:555-566; Brown, et al., (1987) Cell 49:603-612; Figge, et al., (1988)Cell 52:713-722; Deuschle, et al., (1989) Proc. Natl. Acad. Sci. USA86:5400-5404; Fuerst, et al., (1989) Proc. Natl. Acad. Sci. USA86:2549-2553; Deuschle, et al., (1990) Science 248: 480-483; Gossen,(1993) Ph.D. Thesis, University of Heidelberg; Reines, et al., (1993)Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow, et al., (1990) Mol.Cell. Biol. 10:3343-3356; Zambretti, et al., (1992) Proc. Natl. Acad.Sci. USA 89:3952-3956; Baim, et al., (1991) Proc. Natl. Acad. Sci. USA88:5072-5076; Wyborski, et al., (1991) Nucleic Acids Res. 19:4647-4653;Hillenand-Wissman, (1989) Topics Mol. Struc. Biol. 10:143-162;Degenkolb, et al., (1991) Antimicrob. Agents Chemother. 35:1591-1595;Kleinschnidt, et al., (1988) Biochemistry 27:1094-1104; Bonin, (1993)Ph.D. Thesis, University of Heidelberg; Gossen, et al., (1992) Proc.Natl. Acad. Sci. USA 89:5547-5551; Oliva, et al., (1992) Antimicrob.Agents Chemother. 36:913-919; Hlavka, et al., (1985) Handbook ofExperimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin) and Gill,et al., (1988) Nature 334:721-724. Such disclosures are hereinincorporated by reference.

The above list of selectable marker genes is not meant to be limiting.Any selectable marker gene can be used in the embodiments.

Plant Transformation

The methods of the embodiments involve introducing a polypeptide orpolynucleotide into a plant. “Introducing” is as used herein meanspresenting to the plant the polynucleotide or polypeptide in such amanner that the sequence gains access to the interior of a cell of theplant. The methods of the embodiments do not depend on a particularmethod for introducing a polynucleotide or polypeptide into a plant,only that the polynucleotide or polypeptides gains access to theinterior of at least one cell of the plant. Methods for introducingpolynucleotide or polypeptides into plants are known in the artincluding, but not limited to, stable transformation methods, transienttransformation methods, and virus-mediated methods.

“Stable transformation” is as used herein means that the nucleotideconstruct introduced into a plant integrates into the genome of theplant and is capable of being inherited by the progeny thereof.“Transient transformation” as used herein means that a polynucleotide isintroduced into the plant and does not integrate into the genome of theplant or a polypeptide is introduced into a plant. “Plant” as usedherein refers to whole plants, plant organs (e.g., leaves, stems, roots,etc.), seeds, plant cells, propagules, embryos and progeny of the same.Plant cells can be differentiated or undifferentiated (e.g. callus,suspension culture cells, protoplasts, leaf cells, root cells, phloemcells, and pollen).

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Suitablemethods of introducing nucleotide sequences into plant cells andsubsequent insertion into the plant genome include microinjection(Crossway, et al., (1986) Biotechniques 4:320-334), electroporation(Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606),Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and5,981,840), direct gene transfer (Paszkowski, et al., (1984) EMBO J.3:2717-2722) and ballistic particle acceleration (see, for example, U.S.Pat. Nos. 4,945,050; 5,879,918; 5,886,244 and 5,932,782; Tomes, et al.,(1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods,ed. Gamborg and Phillips (Springer-Verlag, Berlin) and McCabe, et al.,(1988) Biotechnology 6:923-926) and Led transformation (WO 00/28058).For potato transformation see, Tu, et al., (1998) Plant MolecularBiology 37:829-838 and Chong, et al., (2000) Transgenic Research9:71-78. Additional transformation procedures can be found inWeissinger, et al., (1988) Ann. Rev. Genet. 22:421-477; Sanford, et al.,(1987) Particulate Science and Technology 5:27-37 (onion); Christou, etal., (1988) Plant Physiol. 87:671-674 (soybean); McCabe, et al., (1988)Biotechnology 6:923-926 (soybean); Finer and McMullen, (1991) In VitroCell Dev. Biol. 27P:175-182 (soybean); Singh, et al., (1998) Theor.Appl. Genet. 96:319-324 (soybean); Datta, et al., (1990) Biotechnology8:736-740 (rice); Klein, et al., (1988) Proc. Natl. Acad. Sci. USA85:4305-4309 (maize); Klein, et al., (1988) Biotechnology 6:559-563(maize); U.S. Pat. Nos. 5,240,855; 5,322,783 and 5,324,646; Klein, etal., (1988) Plant Physiol. 91:440-444 (maize); Fromm, et al., (1990)Biotechnology 8:833-839 (maize); Hooykaas Van Slogteren, et al., (1984)Nature (London) 311:763-764; U.S. Pat. No. 5,736,369 (cereals);Bytebier, et al., (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349(Liliaceae); De Wet, et al., (1985) in The Experimental Manipulation ofOvule Tissues, ed. Chapman, et al., (Longman, N.Y.), pp. 197-209(pollen); Kaeppler, et al., (1990) Plant Cell Reports 9:415-418 andKaeppler, et al., (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin, et al., (1992) Plant Cell4:1495-1505 (electroporation); Li, et al., (1993) Plant Cell Reports12:250-255 and Christou and Ford, (1995) Annals of Botany 75:407-413(rice); Osjoda, et al., (1996) Nature Biotechnology 14:745-750 (maizevia Agrobacterium tumefaciens); all of which are herein incorporated byreference.

In specific embodiments, the sequences of the embodiments can beprovided to a plant using a variety of transient transformation methods.Such transient transformation methods include, but are not limited to,the introduction of the polypeptide or variants and fragments thereofdirectly into the plant or the introduction of the polypeptidetranscript into the plant. Such methods include, for example,microinjection or particle bombardment. See, for example, Crossway, etal., (1986) Mol Gen. Genet. 202:179-185; Nomura, et al., (1986) PlantSci. 44:53-58; Hepler, et al., (1994) Proc. Natl. Acad. Sci. USA91:2176-2180 and Hush, et al., (1994) The Journal of Cell Science107:775-784, all of which are herein incorporated by reference.

Alternatively, the polypeptide polynucleotide can be transientlytransformed into the plant using techniques known in the art. Suchtechniques include viral vector system and the precipitation of thepolynucleotide in a manner that precludes subsequent release of the DNA.Thus, transcription from the particle bound DNA can occur, but thefrequency with which it is released to become integrated into the genomeis greatly reduced. Such methods include the use of particles coatedwith polyethylenimine (PEI; Sigma #P3143).

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site specific recombination system. See,for example, WO 1999/25821, WO 1999/25854, WO 1999/25840, WO 1999/25855and WO 1999/25853, all of which are herein incorporated by reference.Briefly, the polynucleotide of the embodiments can be contained intransfer cassette flanked by two non-identical recombination sites. Thetransfer cassette is introduced into a plant have stably incorporatedinto its genome a target site which is flanked by two non-identicalrecombination sites that correspond to the sites of the transfercassette. An appropriate recombinase is provided and the transfercassette is integrated at the target site. The polynucleotide ofinterest is thereby integrated at a specific chromosomal position in theplant genome.

Plant transformation vectors may be comprised of one or more DNA vectorsneeded for achieving plant transformation. For example, it is a commonpractice in the art to utilize plant transformation vectors that arecomprised of more than one contiguous DNA segment. These vectors areoften referred to in the art as “binary vectors”. Binary vectors as wellas vectors with helper plasmids are most often used forAgrobacterium-mediated transformation, where the size and complexity ofDNA segments needed to achieve efficient transformation is quite large,and it is advantageous to separate functions onto separate DNAmolecules. Binary vectors typically contain a plasmid vector thatcontains the cis-acting sequences required for T-DNA transfer (such asleft border and right border), a selectable marker that is engineered tobe capable of expression in a plant cell, and a “gene of interest” (agene engineered to be capable of expression in a plant cell for whichgeneration of transgenic plants is desired). Also present on thisplasmid vector are sequences required for bacterial replication. Thecis-acting sequences are arranged in a fashion to allow efficienttransfer into plant cells and expression therein. For example, theselectable marker gene and the pesticidal gene are located between theleft and right borders. Often a second plasmid vector contains thetrans-acting factors that mediate T-DNA transfer from Agrobacterium toplant cells. This plasmid often contains the virulence functions (Virgenes) that allow infection of plant cells by Agrobacterium, andtransfer of DNA by cleavage at border sequences and vir-mediated DNAtransfer, as is understood in the art (Hellens and Mullineaux, (2000)Trends in Plant Science 5:446-451). Several types of Agrobacteriumstrains (e.g. LBA4404, GV3101, EHA101, EHA105, etc.) can be used forplant transformation. The second plasmid vector is not necessary fortransforming the plants by other methods such as microprojection,microinjection, electroporation, polyethylene glycol, etc.

In general, plant transformation methods involve transferringheterologous DNA into target plant cells (e.g., immature or matureembryos, suspension cultures, undifferentiated callus, protoplasts,etc.), followed by applying a maximum threshold level of appropriateselection (depending on the selectable marker gene) to recover thetransformed plant cells from a group of untransformed cell mass.Following integration of heterologous foreign DNA into plant cells, onethen applies a maximum threshold level of appropriate selection in themedium to kill the untransformed cells and separate and proliferate theputatively transformed cells that survive from this selection treatmentby transferring regularly to a fresh medium. By continuous passage andchallenge with appropriate selection, one identifies and proliferatesthe cells that are transformed with the plasmid vector. Molecular andbiochemical methods can then be used to confirm the presence of theintegrated heterologous gene of interest into the genome of thetransgenic plant.

Explants are typically transferred to a fresh supply of the same mediumand cultured routinely. Subsequently, the transformed cells aredifferentiated into shoots after placing on regeneration mediumsupplemented with a maximum threshold level of selecting agent. Theshoots are then transferred to a selective rooting medium for recoveringrooted shoot or plantlet. The transgenic plantlet then grows into amature plant and produces fertile seeds (e.g., Hiei, et al., (1994) ThePlant Journal 6:271-282; Ishida, et al., (1996) Nature Biotechnology14:745-750). Explants are typically transferred to a fresh supply of thesame medium and cultured routinely. A general description of thetechniques and methods for generating transgenic plants are found inAyres and Park, (1994) Critical Reviews in Plant Science 13:219-239 andBommineni and Jauhar, (1997) Maydica 42:107-120. Since the transformedmaterial contains many cells; both transformed and non-transformed cellsare present in any piece of subjected target callus or tissue or groupof cells. The ability to kill non-transformed cells and allowtransformed cells to proliferate results in transformed plant cultures.Often, the ability to remove non-transformed cells is a limitation torapid recovery of transformed plant cells and successful generation oftransgenic plants.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick, et al.,(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive or inducible expression ofthe desired phenotypic characteristic identified. Two or moregenerations may be grown to ensure that expression of the desiredphenotypic characteristic is stably maintained and inherited and thenseeds harvested to ensure that expression of the desired phenotypiccharacteristic has been achieved.

The nucleotide sequences of the embodiments may be provided to the plantby contacting the plant with a virus or viral nucleic acids. Generally,such methods involve incorporating the nucleotide construct of interestwithin a viral DNA or RNA molecule. It is recognized that therecombinant proteins of the embodiments may be initially synthesized aspart of a viral polyprotein, which later may be processed by proteolysisin vivo or in vitro to produce the desired polypeptide. It is alsorecognized that such a viral polyprotein, comprising at least a portionof the amino acid sequence of a polypeptide of the embodiments, may havethe desired pesticidal activity. Such viral polyproteins and thenucleotide sequences that encode for them are encompassed by theembodiments. Methods for providing plants with nucleotide constructs andproducing the encoded proteins in the plants, which involve viral DNA orRNA molecules are known in the art. See, for example, U.S. Pat. Nos.5,889,191; 5,889,190; 5,866,785; 5,589,367 and 5,316,931; hereinincorporated by reference.

Methods for transformation of chloroplasts are known in the art. See,for example, Svab, et al., (1990) Proc. Natl. Acad. Sci. USA87:8526-8530; Svab and Maliga, (1993) Proc. Natl. Acad. Sci. USA90:913-917; Svab and Maliga, (1993) EMBO J. 12:601-606. The methodrelies on particle gun delivery of DNA containing a selectable markerand targeting of the DNA to the plastid genome through homologousrecombination. Additionally, plastid transformation can be accomplishedby transactivation of a silent plastid-borne transgene bytissue-preferred expression of a nuclear encoded and plastid directedRNA polymerase. Such a system has been reported in McBride, et al.,(1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.

The embodiments further relate to plant propagating material of atransformed plant of the embodiments including, but not limited to,seeds, tubers, corms, bulbs, leaves and cuttings of roots and shoots.

Plant Species Capable of being Transformed and Expressing anInsecticidal Protein

The embodiments may be used for transformation of any plant species,including, but not limited to, monocots and dicots. Examples of plantsof interest include, but are not limited to, corn (Zea mays), Brassicasp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassicaspecies useful as sources of seed oil, alfalfa (Medicago sativa), rice(Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghumvulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet(Panicum miliaceum), foxtail millet (Setaria italica), finger millet(Eleusine coracana)), sunflower (Helianthus annuus), safflower(Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycinemax), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts(Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum),sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee(Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley,vegetables, ornamentals, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pukherrima), and chrysanthemum. Conifers that may be employedin practicing the embodiments include, for example, pines such asloblolly pine (Pinus taeda), slash pine (Pinus elliottii), ponderosapine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Montereypine (Pinus radiata); Douglas fir (Pseudotsuga menziesii); Westernhemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood(Sequoia sempervirens); true first such as silver fir (Abies amabilis)and balsam fir (Abies balsamea); and cedars such as Western red cedar(Thuja plicata) and Alaska yellow cedar (Chamaecyparis nootkatensis).Plants of the embodiments include crop plants (for example, corn,alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut,sorghum, wheat, millet, tobacco, etc.), such as corn and soybean plants.

Turf grasses include, but are not limited to: annual bluegrass (Poaannua); annual ryegrass (Lolium multiflorum); Canada bluegrass (Poacompressa); Chewing's fescue (Festuca rubra); colonial bentgrass(Agrostis tenuis); creeping bentgrass (Agrostis palustris); crestedwheatgrass (Agropyron desertorum); fairway wheatgrass (Agropyroncristadtum); hard fescue (Festuca longifolia); Kentucky bluegrass (Poapratensis); orchardgrass (Dactylis glomerata); perennial ryegrass(Lolium perenne); red fescue (Festuca rubra); redtop (Agrostis alba);rough bluegrass (Paa trivialis); sheep fescue (Festuca ovina); smoothbromegrass (Bromus inermis); tall fescue (Festuca arundinacea); timothy(Phleum pratense); velvet bentgrass (Agrostis canina); weepingalkaligrass (Puccinellia distans); western wheatgrass (Agropyronsmithi); Bermuda grass (Cynodon spp.); St. Augustine grass (Stenotaphrumsecundatum); zoysia grass (Zoysia spp.); Bahia grass (Paspalum notatum);carpet grass (Axonopus aifinis); centipede grass (Eremochloaophiuroides); kikuyu grass (Pennisetum clandesinum); seashore paspalum(Paspalum vaginatum); blue gramma (Bouteloua gracilis); buffalo grass(Buchloe dactyloids); sideoats gramma (Bouteloua curtipendula).

Plants of interest include cereals, grain plants that provide seeds ofinterest, oil-seed plants, and leguminous plants. Seeds of interestinclude grain seeds, such as corn, wheat, barley, rice, sorghum, rye,millet, etc. Oil-seed plants include cotton, soybean, safflower,sunflower, Brassica, maize, alfalfa, palm, coconut, flax, castor, olive,etc. Leguminous plants include beans and peas. Beans include guar,locust bean, fenugreek, soybean, garden beans, cowpea, mung bean, limabean, fava bean, lentils, chickpea, etc.

Evaluation of Transformation

Following introduction of heterologous foreign DNA into plant cells, thetransformation or integration of a heterologous gene into the plantgenome is confirmed by various methods such as analysis of nucleicacids, proteins and metabolites associated with the integrated gene.

PCR analysis is a rapid method to screen transformed cells, tissue orshoots for the presence of an incorporated gene at the earlier stagebefore transplanting into the soil (Sambrook and Russell, (2001)Molecular Cloning: A Laboratory Manual. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.). PCR is carried out usingoligonucleotide primers specific to the gene of interest orAgrobacterium vector background, etc.

Plant transformation may be confirmed by Southern blot analysis ofgenomic DNA (Sambrook and Russell, (2001) supra). In general, total DNAis extracted from the transformant, digested with appropriaterestriction enzymes, fractionated in an agarose gel and transferred to anitrocellulose or nylon membrane. The membrane or “blot” is then probedwith, for example, radiolabeled 32P target DNA fragment to confirm theintegration of an introduced gene into the plant genome according tostandard techniques (Sambrook and Russell, (2001) supra).

In Northern blot analysis, RNA is isolated from specific tissues oftransformant, fractionated in a formaldehyde agarose gel, and blottedonto a nylon filter according to standard procedures that are routinelyused in the art (Sambrook and Russell, (2001) supra). Expression of RNAencoded by the pesticidal gene is then tested by hybridizing the filterto a radioactive probe derived from a pesticidal gene, by methods knownin the art (Sambrook and Russell, (2001) supra).

Western blot, biochemical assays and the like may be carried out on thetransgenic plants to confirm the presence of protein encoded by thepesticidal gene by standard procedures (Sambrook and Russell, 2001,supra) using antibodies that bind to one or more epitopes present on thetaught insecticidal proteins.

Stacking of Transgenic Traits in a Plant

Transgenic plants may comprise a stack of one or more insecticidalpolynucleotides disclosed herein with one or more additionalpolynucleotides resulting in the production or suppression of multiplepolypeptide sequences.

Transgenic plants comprising stacks of polynucleotide sequences can beobtained by either or both of traditional breeding methods or throughgenetic engineering methods. These methods include, but are not limitedto, breeding individual lines each comprising a polynucleotide ofinterest, transforming a transgenic plant comprising a gene disclosedherein with a subsequent gene and co-transformation of genes into asingle plant cell.

As used herein, the term “stacked” includes having the multiple traitspresent in the same plant (i.e., both traits are incorporated into thenuclear genome, one trait is incorporated into the nuclear genome andone trait is incorporated into the genome of a plastid, or both traitsare incorporated into the genome of a plastid). In one non-limitingexample, “stacked traits” comprise a molecular stack where the sequencesare physically adjacent to each other. A trait, as used herein, refersto the phenotype derived from a particular sequence or groups ofsequences. Co-transformation of genes can be carried out using singletransformation vectors comprising multiple genes or genes carriedseparately on multiple vectors. If the sequences are stacked bygenetically transforming the plants, the polynucleotide sequences ofinterest can be combined at any time and in any order. The traits can beintroduced simultaneously in a co-transformation protocol with thepolynucleotides of interest provided by any combination oftransformation cassettes. For example, if two sequences will beintroduced, the two sequences can be contained in separatetransformation cassettes (trans) or contained on the same transformationcassette (cis). Expression of the sequences can be driven by the samepromoter or by different promoters. In certain cases, it may bedesirable to introduce a transformation cassette that will suppress theexpression of the polynucleotide of interest. This may be combined withany combination of other suppression cassettes or overexpressioncassettes to generate the desired combination of traits in the plant. Itis further recognized that polynucleotide sequences can be stacked at adesired genomic location using a site-specific recombination system.See, for example, WO 1999/25821, WO 1999/25854, WO 1999/25840, WO1999/25855 and WO 1999/25853, all of which are herein incorporated byreference.

In some embodiments, the polynucleotides encoding the pesticidalproteins disclosed herein, alone or stacked with one or more additionalinsect resistance traits, can be stacked with one or more additionalinput traits (e.g., herbicide resistance, fungal resistance, virusresistance, stress tolerance, disease resistance, male sterility, stalkstrength, and the like) or output traits (e.g., increased yield,modified starches, improved oil profile, balanced amino acids, highlysine or methionine, increased digestibility, improved fiber quality,drought resistance, and the like). Thus, the polynucleotide embodimentscan be used to provide a complete agronomic package of improved cropquality with the ability to flexibly and cost effectively control anynumber of agronomic pests.

Transgenes useful for stacking include other pesticidal proteins, suchas: Monalysin, PIP, Cry, Cyt, Vip, TC, and any combination thereof.These pesticidal proteins have been set forth in great detail in earliersections of the specification.

Other transgenes useful for stacking with the taught pesticidal proteinsinclude genes encoding for: plant disease resistance, insect specifichormones or pheromones, antifungal activity, and nematicidal activity.

Transgenes that confer resistance to an herbicide can also be stackedwith the taught pesticidal proteins, including (non-limiting class of 9herbicidal classes below):

(1) A polynucleotide encoding resistance to an herbicide that inhibitsthe growing point or meristem, such as an imidazolinone or asulfonylurea. Exemplary genes in this category code for mutant ALS andAHAS enzyme as described, for example, by Lee, et al., (1988) EMBO J.7:1241 and Miki, et al., (1990) Theor. Appl. Genet. 80:449,respectively. See also, U.S. Pat. Nos. 5,605,011; 5,013,659; 5,141,870;5,767,361; 5,731,180; 5,304,732; 4,761,373; 5,331,107; 5,928,937 and5,378,824; U.S. patent application Ser. No. 11/683,737 and InternationalPublication WO 1996/33270.

(2) A polynucleotide encoding a protein for resistance to Glyphosate(resistance imparted by mutant 5-enolpyruvl-3-phosphikimate synthase(EPSPS) and aroA genes, respectively) and other phosphono compounds suchas glufosinate (phosphinothricin acetyl transferase (PAT) andStreptomyces hygroscopicus phosphinothricin acetyl transferase (bar)genes), and pyridinoxy or phenoxy proprionic acids and cyclohexones(ACCase inhibitor-encoding genes). See, for example, U.S. Pat. No.4,940,835 to Shah, et al., which discloses the nucleotide sequence of aform of EPSPS which can confer glyphosate resistance. U.S. Pat. No.5,627,061 to Barry, et al., also describes genes encoding EPSPS enzymes.See also, U.S. Pat. Nos. 6,566,587; 6,338,961; 6,248,876 B1; U.S. Pat.Nos. 6,040,497; 5,804,425; 5,633,435; 5,145, 783; 4,971,908; 5,312,910;5,188,642; 5,094,945, 4,940, 835; 5,866,775; 6,225,114 B1; U.S. Pat.Nos. 6,130,366; 5,310,667; 4,535,060; 4,769,061; 5,633,448; 5,510,471;Re. 36,449; RE 37,287 E and 5,491,288 and International Publications EP1173580; WO 2001/66704; EP 1173581 and EP 1173582, which areincorporated herein by reference for this purpose. Glyphosate resistanceis also imparted to plants that express a gene encoding a glyphosateoxido-reductase enzyme as described more fully in U.S. Pat. Nos.5,776,760 and 5,463,175, which are incorporated herein by reference forthis purpose. In addition, glyphosate resistance can be imparted toplants by the over expression of genes encoding glyphosateN-acetyltransferase. See, for example, U.S. Pat. Nos. 7,462,481;7,405,074 and US Patent Application Publication Number US 2008/0234130.A DNA molecule encoding a mutant aroA gene can be obtained under ATCC®Accession Number 39256, and the nucleotide sequence of the mutant geneis disclosed in U.S. Pat. No. 4,769,061 to Comai. EP Application Number0 333 033 to Kumada, et al., and U.S. Pat. No. 4,975,374 to Goodman, etal., disclose nucleotide sequences of glutamine synthetase genes whichconfer resistance to herbicides such as L-phosphinothricin. Thenucleotide sequence of a phosphinothricin-acetyl-transferase gene isprovided in EP Application Numbers 0 242 246 and 0 242 236 to Leemans,et al., De Greef, et al., (1989) Biol Technology 7:61, describe theproduction of transgenic plants that express chimeric bar genes codingfor phosphinothricin acetyl transferase activity. See also, U.S. Pat.Nos. 5,969,213; 5,489,520; 5,550,318; 5,874,265; 5,919,675; 5,561,236;5,648,477; 5,646,024; 6,177,616 B1 and 5,879, 903, which areincorporated herein by reference for this purpose. Exemplary genesconferring resistance to phenoxy propionic acids and cyclohexanes, suchas sethoxydim and haloxyfop, are the Acc1-S1, Acc1-S2 and Acc1-S3 genesdescribed by Marshall, et al., (1992) Theor. Appl. Genet. 83:435.

(3) A polynucleotide encoding a protein for resistance to herbicide thatinhibits photosynthesis, such as a triazine (psbA and gs+ genes) and abenzonitrile (nitrilase gene). Przibilla, et al., (1991) Plant Cell3:169, describe the transformation of Chlamydomonas with plasmidsencoding mutant psbA genes. Nucleotide sequences for nitrilase genes aredisclosed in U.S. Pat. No. 4,810,648 to Stalker and DNA moleculescontaining these genes are available under ATCC® Accession Numbers53435, 67441 and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes, et al., (1992) Biochem.J. 285:173.

(4) A polynucleotide encoding a protein for resistance to Acetohydroxyacid synthase, which has been found to make plants that express thisenzyme resistant to multiple types of herbicides, has been introducedinto a variety of plants (see, e.g., Hattori, et al., (1995) Mol. Gen.Genet. 246:419). Other genes that confer resistance to herbicidesinclude: a gene encoding a chimeric protein of rat cytochrome P4507A1and yeast NADPH-cytochrome P450 oxidoreductase (Shiota, et al., (1994)Plant Physiol. 106:1 7), genes for glutathione reductase and superoxidedismutase (Aono, et al., (1995) Plant Cell Physiol. 36:1687) and genesfor various phosphotransferases (Datta, et al., (1992) Plant Mol. Biol.20:619).

(5) A polynucleotide encoding resistance to a herbicide targetingProtoporphyrinogen oxidase (protox) which is necessary for theproduction of chlorophyll. The protox enzyme serves as the target for avariety of herbicidal compounds. These herbicides also inhibit growth ofall the different species of plants present, causing their totaldestruction. The development of plants containing altered protoxactivity which are resistant to these herbicides are described in U.S.Pat. Nos. 6,288,306 B1; 6,282,837 B1 and 5,767,373 and InternationalPublication WO 2001/12825.

(6) The aad-1 gene (originally from Sphingobium herbicidovorans) encodesthe aryloxyalkanoate dioxygenase (AAD-1) protein. The trait conferstolerance to 2,4-dichlorophenoxyacetic acid and aryloxyphenoxypropionate(commonly referred to as “fop” herbicides such as quizalofop)herbicides. The aad-1 gene, itself, for herbicide-tolerance in plantswas first disclosed in WO 2005/107437 (see also, US 2009/0093366). Theaad-12 gene, derived from Delftia acidovorans, which encodes thearyloxyalkanoate dioxygenase (AAD-12) protein that confers tolerance to2,4-dichlorophenoxyacetic acid and pyridyloxyacetate herbicides bydeactivating several herbicides with an aryloxyalkanoate moiety,including phenoxy auxin (e.g., 2,4-D, MCPA), as well as pyridyloxyauxins (e.g., fluroxypyr, triclopyr).

(7) A polynucleotide encoding a herbicide resistant dicambamonooxygenase disclosed in US Patent Application Publication2003/0135879 for imparting dicamba tolerance;

(8) A polynucleotide molecule encoding bromoxynil nitrilase (Bxn)disclosed in U.S. Pat. No. 4,810,648 for imparting bromoxynil tolerance;

(9) A polynucleotide molecule encoding phytoene (crtl) described inMisawa, et al., (1993) Plant J. 4:833-840 and in Misawa, et al., (1994)Plant J. 6:481-489 for norflurazon tolerance.

Transgenes that confer or contribute to an altered grain characteristiccan also be stacked with the taught pesticidal proteins, including(non-limiting class below relating to altered fatty acids in grain): (1)Down-regulation of stearoyl-ACP to increase stearic acid content of theplant. See, Knultzon, et al., (1992) Proc. Natl. Acad. Sci. USA 89:2624and WO 1999/64579 (Genes to Alter Lipid Profiles in Corn). (2) Elevatingoleic acid via FAD-2 gene modification and/or decreasing linolenic acidvia FAD-3 gene modification (see, U.S. Pat. Nos. 6,063,947; 6,323,392;6,372,965 and WO 1993/11245). (3) Altering conjugated linolenic orlinoleic acid content, such as in WO 2001/12800. (4) Altering LEC1, AGP,Dekl, Superall, mil ps, various Ipa genes such as Ipa1, Ipa3, hpt orhggt. For example, see, WO 2002/42424, WO 1998/22604, WO 2003/011015, WO2002/057439, WO 2003/011015, U.S. Pat. Nos. 6,423,886, 6,197,561,6,825,397 and US Patent Application Publication Numbers US 2003/0079247,US 2003/0204870 and Rivera-Madrid, et al., (1995) Proc. Natl. Acad. Sci.USA 92:5620-5624. (5) Genes encoding delta-8 desaturase for makinglong-chain polyunsaturated fatty acids (U.S. Pat. Nos. 8,058,571 and8,338,152), delta-9 desaturase for lowering saturated fats (U.S. Pat.No. 8,063,269), Primula A6-desaturase for improving omega-3 fatty acidprofiles. (6) Isolated nucleic acids and proteins associated with lipidand sugar metabolism regulation, in particular, lipid metabolism protein(LMP) used in methods of producing transgenic plants and modulatinglevels of seed storage compounds including lipids, fatty acids, starchesor seed storage proteins and use in methods of modulating the seed size,seed number, seed weights, root length and leaf size of plants (EP2404499). (7) Altering expression of a High-Level Expression ofSugar-Inducible 2 (HSI2) protein in the plant to increase or decreaseexpression of HSI2 in the plant. Increasing expression of HSI2 increasesoil content while decreasing expression of HSI2 decreases abscisic acidsensitivity and/or increases drought resistance (US Patent ApplicationPublication Number 2012/0066794). (8) Expression of cytochrome b5 (Cb5)alone or with FAD2 to modulate oil content in plant seed, particularlyto increase the levels of omega-3 fatty acids and improve the ratio ofomega-6 to omega-3 fatty acids (US Patent Application Publication Number2011/0191904). (9) Nucleic acid molecules encoding wrinkled1-likepolypeptides for modulating sugar metabolism (U.S. Pat. No. 8,217,223).

Transgenes that confer or contribute to an altered grain characteristiccan also be stacked with the taught pesticidal proteins, including(non-limiting class below relating to altered phosphorus content ingrain): (1) Introduction of a phytase encoding gene would enhancebreakdown of phytate, adding more free phosphate to the transformedplant. For example, see, Van Hartingsveldt, et al., (1993) Gene 127:87,for a disclosure of the nucleotide sequence of an Aspergillus nigerphytase gene. (2) Modulating a gene that reduces phytate content. Inmaize, this, for example, could be accomplished, by cloning and thenreintroducing DNA associated with one or more of the alleles, such asthe LPA alleles, identified in maize mutants characterized by low levelsof phytic acid, such as in WO 2005/113778 and/or by altering inositolkinase activity as in WO 2002/059324, US Patent Application PublicationNumber 2003/0009011, WO 2003/027243, US Patent Application PublicationNumber 2003/0079247, WO 1999/05298, U.S. Pat. Nos. 6,197,561, 6,291,224,6,391,348, WO 2002/059324, US Patent Application Publication Number2003/0079247, WO 1998/45448, WO 1999/55882, WO 2001/04147.

Transgenes that confer or contribute to an altered grain characteristiccan also be stacked with the taught pesticidal proteins, including(non-limiting class below relating to altered carbohydrate content ingrain): (1) altering a gene for an enzyme that affects the branchingpattern of starch or, a gene altering thioredoxin such as NTR and/or TRX(see, U.S. Pat. No. 6,531,648. which is incorporated by reference forthis purpose) and/or a gamma zein knock out or mutant such as cs27 orTUSC27 or en27 (see, U.S. Pat. No. 6,858,778 and US Patent ApplicationPublication Number 2005/0160488, US Patent Application PublicationNumber 2005/0204418, which are incorporated by reference for thispurpose). See, Shiroza, et al., (1988) J. Bacteriol. 170:810 (nucleotidesequence of Streptococcus mutant fructosyltransferase gene), Steinmetz,et al., (1985) Mol. Gen. Genet. 200:220 (nucleotide sequence of Bacillussubtilis levansucrase gene), Pen, et al., (1992) Biotechnology 10:292(production of transgenic plants that express Bacillus licheniformisalpha-amylase), Elliot, et al., (1993) Plant Molec. Biol. 21:515(nucleotide sequences of tomato invertase genes), Segaard, et al.,(1993) J. Biol. Chem. 268:22480 (site-directed mutagenesis of barleyalpha-amylase gene) and Fisher, et al., (1993) Plant Physiol. 102:1045(maize endosperm starch branching enzyme II), WO 1999/10498 (improveddigestibility and/or starch extraction through modification ofUDP-D-xylose 4-epimerase, Fragile 1 and 2, Refl, HCHL, C4H), U.S. Pat.No. 6,232,529 (method of producing high oil seed by modification ofstarch levels (AGP)). The fatty acid modification genes mentioned hereinmay also be used to affect starch content and/or composition through theinterrelationship of the starch and oil pathways.

Transgenes that confer or contribute to an altered grain characteristiccan also be stacked with the taught pesticidal proteins, including(non-limiting class below relating to altered antioxidant content ingrain): (1) alteration of tocopherol or tocotrienols. For example, see,U.S. Pat. No. 6,787,683, US Patent Application Publication Number2004/0034886 and WO 2000/68393 involving the manipulation of antioxidantlevels and WO 2003/082899 through alteration of a homogentisategeranylgeranyl transferase (hggt).

Transgenes that confer or contribute to an altered grain characteristiccan also be stacked with the taught pesticidal proteins, including(non-limiting class below relating to altered essential amino acidcontent in grain): (1) For example, see, U.S. Pat. No. 6,127,600 (methodof increasing accumulation of essential amino acids in seeds), U.S. Pat.No. 6,080,913 (binary methods of increasing accumulation of essentialamino acids in seeds), U.S. Pat. No. 5,990,389 (high lysine), WO1999/40209 (alteration of amino acid compositions in seeds), WO1999/29882 (methods for altering amino acid content of proteins), U.S.Pat. No. 5,850,016 (alteration of amino acid compositions in seeds), WO1998/20133 (proteins with enhanced levels of essential amino acids),U.S. Pat. No. 5,885,802 (high methionine), U.S. Pat. No. 5,885,801 (highthreonine), U.S. Pat. No. 6,664,445 (plant amino acid biosyntheticenzymes), U.S. Pat. No. 6,459,019 (increased lysine and threonine), U.S.Pat. No. 6,441,274 (plant tryptophan synthase beta subunit), U.S. Pat.No. 6,346,403 (methionine metabolic enzymes), U.S. Pat. No. 5,939,599(high sulfur), U.S. Pat. No. 5,912,414 (increased methionine), WO1998/56935 (plant amino acid biosynthetic enzymes), WO 1998/45458(engineered seed protein having higher percentage of essential aminoacids), WO 1998/42831 (increased lysine), U.S. Pat. No. 5,633,436(increasing sulfur amino acid content), U.S. Pat. No. 5,559,223(synthetic storage proteins with defined structure containingprogrammable levels of essential amino acids for improvement of thenutritional value of plants), WO 1996/01905 (increased threonine), WO1995/15392 (increased lysine), US Patent Application Publication Number2003/0163838, US Patent Application Publication Number 2003/0150014, USPatent Application Publication Number 2004/0068767, U.S. Pat. No.6,803,498, WO 2001/79516.

Transgenes that confer or contribute to male sterility can also bestacked with the taught pesticidal proteins. Transgenes that create asite for site specific DNA integration can also be stacked with thetaught pesticidal proteins.

Transgenes that affect abiotic stress resistance of a crop plant canalso be stacked with the taught pesticidal proteins, including, but notlimited to: flowering, ear and seed development, enhancement of nitrogenutilization efficiency, altered nitrogen responsiveness, droughtresistance or tolerance, cold resistance or tolerance and saltresistance or tolerance and increased yield under stress. Furtherexamples of abiotic stress resistance genes that can be stacked with thetaught pesticidal proteins, include: (1) WO 2000/73475 where water useefficiency is altered through alteration of malate; U.S. Pat. Nos.5,892,009, 5,965,705, 5,929,305, 5,891,859, 6,417,428, 6,664,446,6,706,866, 6,717,034, 6,801,104, WO 2000/060089, WO 2001/026459, WO2001/035725, WO 2001/034726, WO 2001/035727, WO 2001/036444, WO2001/036597, WO 2001/036598, WO 2002/015675, WO 2002/017430, WO2002/077185, WO 2002/079403, WO 2003/013227, WO 2003/013228, WO2003/014327, WO 2004/031349, WO 2004/076638, WO 199809521. (2) WO199938977 describing genes, including CBF genes and transcriptionfactors effective in mitigating the negative effects of freezing, highsalinity and drought on plants, as well as conferring other positiveeffects on plant phenotype. (3) US Patent Application Publication Number2004/0148654 and WO 2001/36596 where abscisic acid is altered in plantsresulting in improved plant phenotype such as increased yield and/orincreased tolerance to abiotic stress. (4) WO 2000/006341, WO2004/090143, U.S. Pat. Nos. 7,531,723 and 6,992,237 where cytokininexpression is modified resulting in plants with increased stresstolerance, such as drought tolerance, and/or increased yield. Also see,WO 2002/02776, WO 2003/052063, JP 2002/281975, U.S. Pat. No. 6,084,153,WO 2001/64898, U.S. Pat. Nos. 6,177,275 and 6,107,547 (enhancement ofnitrogen utilization and altered nitrogen responsiveness). (5) Forethylene alteration, see, US Patent Application Publication Number2004/0128719, US Patent Application Publication Number 2003/0166197 andWO 2000/32761. (6) For plant transcription factors or transcriptionalregulators of abiotic stress, see, e.g., US Patent ApplicationPublication Number 2004/0098764 or US Patent Application PublicationNumber 2004/0078852. (7) Genes that increase expression of vacuolarpyrophosphatase such as AVP1 (U.S. Pat. No. 8,058,515) for increasedyield; nucleic acid encoding a HSF A4 or a HSFA5 (Heat Shock Factor ofthe class A4 or A5) polypeptides, an oligopeptide transporter protein(OPT4-like) polypeptide; a plastochron2-like (PLA2-like) polypeptide ora Wuschel related homeobox I-like (WOX1-like) polypeptide (U. PatentApplication Publication Number US 2011/0283420). (8) Down regulation ofpolynucleotides encoding poly (ADP-ribose) polymerase (PARP) proteins tomodulate programmed cell death (U.S. Pat. No. 8,058,510) for increasedvigor. (9) Polynucleotide encoding DTP21 polypeptides for conferringdrought resistance (US Patent Application Publication Number US2011/0277181). (10) Nucleotide sequences encoding ACC Synthase 3 (ACS3)proteins for modulating development, modulating response to stress, andmodulating stress tolerance (US Patent Application Publication Number US2010/0287669). (11) Polynucleotides that encode proteins that confer adrought tolerance phenotype (DTP) for conferring drought resistance (WO2012/058528). (12) Tocopherol cyclase (TC) genes for conferring droughtand salt tolerance (US Patent Application Publication Number2012/0272352). (13) CAAX amino terminal family proteins for stresstolerance (U.S. Pat. No. 8,338,661). (14) Mutations in the SAL1 encodinggene have increased stress tolerance, including increased droughtresistant (US Patent Application Publication Number 2010/0257633). (15)Expression of a nucleic acid sequence encoding a polypeptide selectedfrom the group consisting of: GRF polypeptide, RAA1-like polypeptide,SYR polypeptide, ARKL polypeptide, and YTP polypeptide increasingyield-related traits (US Patent Application Publication Number2011/0061133). (16) Modulating expression in a plant of a nucleic acidencoding a Class III Trehalose Phosphate Phosphatase (TPP) polypeptidefor enhancing yield-related traits in plants, particularly increasingseed yield (US Patent Application Publication Number 2010/0024067). (17)Expression of a nucleic acid sequence encoding a Drought TolerantPhenotype (DTP6) polypeptide, specifically AT-DTP6 of US PatentApplication Publication Number 2014/0223595.

Other genes and transcription factors that affect plant growth andagronomic traits such as yield, flowering, plant growth and/or plantstructure, can be introduced or introgressed into plants, see e.g., WO1997/49811 (LHY), WO 1998/56918 (ESD4), WO 1997/10339 and U.S. Pat. No.6,573,430 (TFL), U.S. Pat. No. 6,713,663 (FT), WO 1996/14414 (CON), WO1996/38560, WO 2001/21822 (VRN1), WO 2000/44918 (VRN2), WO 1999/49064(GI), WO 2000/46358 (FRI), WO 1997/29123, U.S. Pat. Nos. 6,794,560,6,307,126 (GAI), WO 1999/09174 (D8 and Rht) and WO 2004/076638 and WO2004/031349 (transcription factors).

Transgenes that confer increased yield to a crop plant can also bestacked with the taught pesticidal proteins, for example: (1) atransgenic crop plant transformed by a 1-AminoCyclopropane-1-CarboxylateDeaminase-like Polypeptide (ACCDP) coding nucleic acid, whereinexpression of the nucleic acid sequence in the crop plant results in theplant's increased root growth, and/or increased yield, and/or increasedtolerance to environmental stress as compared to a wild type variety ofthe plant (U.S. Pat. No. 8,097,769). (2) overexpression of maize zincfinger protein gene (Zm-ZFP1) using a seed preferred promoter has beenshown to enhance plant growth, increase kernel number and total kernelweight per plant (US Patent Application Publication Number2012/0079623). (3) Constitutive overexpression of maize lateral organboundaries (LOB) domain protein (Zm-LOBDP1) has been shown to increasekernel number and total kernel weight per plant (US Patent ApplicationPublication Number 2012/0079622). (4) Enhancing yield-related traits inplants by modulating expression in a plant of a nucleic acid encoding aVIM1 (Variant in Methylation 1)-like polypeptide or a VTC2-like(GDP-L-galactose phosphorylase) polypeptide or a DUF1685 polypeptide oran ARF6-like (Auxin Responsive Factor) polypeptide (WO 2012/038893). (5)Modulating expression in a plant of a nucleic acid encoding a Ste20-likepolypeptide or a homologue thereof gives plants having increased yieldrelative to control plants (EP 2431472). (6) Genes encoding nucleosidediphosphatase kinase (NDK) polypeptides and homologs thereof formodifying the plant's root architecture (US Patent ApplicationPublication Number 2009/0064373).

In some aspects, the pesticidal proteins can be stacked with any genetictrait that has received regulatory approval. A non-exhaustive list ofsuch traits can be found in Table 4A-4F of US 2016/0366891 A1, which isincorporated herein by reference. Furthermore, the taught novelinsecticidal proteins taught herein can be stacked or combined with anygenetic trait from the following Tables B-G listed below.

TABLE B Rice Traits That Can Be Combined With the Insecticidal ProteinsOryza sativa Rice Event Company Description CL121, CL141, CFX51 BASFInc. Tolerance to the imidazolinone herbicide, imazethapyr, induced bychemical mutagenesis of the acetolactate synthase (ALS) enzyme usingethyl methanesulfonate (EMS). IMINTA-1, IMINTA-4 BASF Inc. Tolerance toimidazolinone herbicides induced by chemical mutagenesis of theacetolactate synthase (ALS) enzyme using sodium azide. LLRICE06,LLRICE62 Aventis CropScience Glufosinate ammonium herbicide tolerantrice produced by inserting a modified phosphinothricin acetyltransferase(PAT) encoding gene from the soil bacterium Streptomyces hygroscopicus).LLRICE601 Bayer CropScience (Aventis Glufosinate ammonium herbicideCropScience(AgrEvo)) tolerant rice produced by inserting a modifiedphosphinothricin acetyltransferase (PAT) encoding gene from the soilbacterium Streptomyces hygroscopicus). PWC16 BASF Inc. Tolerance to theimidazolinone herbicide, imazethapyr, induced by chemical mutagenesis ofthe acetolactate synthase (ALS) enzyme using ethyl methanesulfonate(EMS).

TABLE C Alfalfa Traits That Can Be Combined With the InsecticidalProteins Medicago sativa Alfalfa Event Company Description J101, J163Monsanto Company and Glyphosate herbicide tolerant Forage Geneticsalfalfa (lucerne) produced by International inserting a gene encodingthe enzyme 5-enolypyruvylshikimate- 3-phosphate synthase (EPSPS) fromthe CP4 strain of Agrobacterium tumefaciens.

TABLE D Wheat Traits That Can Be Combined With the Insecticidal ProteinsTriticum aestivum Wheat Event Company Description AP205CL BASF Inc.Selection for a mutagenized version of the enzyme acetohydroxyacidsynthase (AHAS), also known as acetolactate synthase (ALS) oracetolactate pyruvate-lyase. AP602CL BASF Inc. Selection for amutagenized version of the enzyme acetohydroxyacid synthase (AHAS), alsoknown as acetolactate synthase (ALS) or acetolactate pyruvate-lyase.BW255-2, BW238-3 BASF Inc. Selection for a mutagenized version of theenzyme acetohydroxyacid synthase (AHAS), also known as acetolactatesynthase (ALS) or acetolactate pyruvate-lyase. BW7 BASF Inc. Toleranceto imidazolinone herbicides induced by chemical mutagenesis of theacetohydroxyacid synthase (AHAS) gene using sodium azide. MON71800Monsanto Company Glyphosate tolerant wheat variety produced by insertinga modified 5- enolpyruvylshikimate-3-phosphate synthase (EPSPS) encodinggene from the soil bacterium Agrobacterium tumefaciens, strain CP4.SWP965001 Cyanamid Crop Selection for a mutagenized version Protectionof the enzyme acetohydroxyacid synthase (AHAS), also known asacetolactate synthase (ALS) or acetolactate pyruvate-lyase. Teal 11ABASF Inc. Selection for a mutagenized version of the enzymeacetohydroxyacid synthase (AHAS), also known as acetolactate synthase(ALS) or acetolactate pyruvate-lyase.

TABLE E Sunflower Traits That Can Be Combined With the InsecticidalProteins Helianthus annuus Sunflower Event Company Description X81359BASF Inc. Tolerance to imidazolinone herbicides by selection of anaturally occurring mutant.

TABLE F Soybean Traits That Can Be Combined With the InsecticidalProteins Glycine max L. Soybean Event Company Description A2704-12,A2704-21, Bayer CropScience Glufosinate ammonium herbicide A5547-35(Aventis CropScience tolerant soybean produced by (AgrEvo)) inserting amodified phosphinothricin acetyltransferase (PAT) encoding gene from thesoil bacterium Streptomyces viridochromogenes. A5547-127 BayerCropScience Glufosinate ammonium herbicide (Aventis CropScience tolerantsoybean produced by (AgrEvo)) inserting a modified phosphinothricinacetyltransferase (PAT) encoding gene from the soil bacteriumStreptomyces viridochromogenes. BPS-CV127-9 BASF Inc. The introducedcsr1-2 gene from Arabidopsis thaliana encodes an acetohydroxyacidsynthase protein that confers tolerance to imidazolinone herbicides dueto a point mutation that results in a single amino acid substitution inwhich the serine residue at position 653 is replaced by asparagine(S653N). DP-305423 Pioneer Hi-Bred High oleic acid soybean producedInternational Inc. by inserting additional copies of a portion of theomega 6 desaturase encoding gene, gm-fad2-1 resulting in silencing ofthe endogenous omega-6 desaturase gene (FAD2-1). DP356043 PioneerHi-Bred Soybean event with two herbicide International Inc. tolerancegenes: glyphosate N- acetlytransferase, which detoxifies glyphosate, anda modified acetolactate synthase (ALS) gene which is tolerant toALS-inhibiting herbicides. G94-1, G94-19, G168 DuPont Canada High oleicacid soybean produced Agricultural Products by inserting a second copyof the fatty acid desaturase (Gm Fad2-1) encoding gene from soybean,which resulted in “silencing” of the endogenous host gene. GTS 40-3-2Monsanto Company Glyphosate tolerant soybean variety produced byinserting a modified 5- enolpyruvylshikimate-3-phosphate synthase(EPSPS) encoding gene from the soil bacterium Agrobacterium tumefaciens.GU262 Bayer CropScience Glufosinate ammonium herbicide (Aventis tolerantsoybean produced by CropScience(AgrEvo)) inserting a modifiedphosphinothricin acetyltransferase (PAT) encoding gene from the soilbacterium Streptomyces viridochromogenes. MON87701 Monsanto CompanyResistance to Lepidopteran pests of soybean including velvetbeancaterpillar (Anticarsia gemmatalis) and soybean looper (Pseudoplusiaincludens). MON87701 × Monsanto Company Glyphosate herbicide toleranceMON89788 through expression of the EPSPS encoding gene from A.tumefaciens strain CP4, and resistance to Lepidopteran pests of soybeanincluding velvetbean caterpillar (Anticarsia gemmatalis) and soybeanlooper (Pseudoplusia includens) via expression of the Cry1Ac encodinggene from B. thuringiensis. MON89788 Monsanto CompanyGlyphosate-tolerant soybean produced by inserting a modified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding aroA (epsps)gene from Agrobacterium tumefaciens CP4. OT96-15 Agriculture & Agri-FoodLow linolenic acid soybean Canada produced through traditional cross-breeding to incorporate the novel trait from a naturally occurring fan1gene mutant that was selected for low linolenic acid. W62, W98 BayerCropScience Glufosinate ammonium herbicide (Aventis tolerant soybeanproduced by CropScience(AgrEvo)) inserting a modified phosphinothricinacetyltransferase (PAT) encoding gene from the soil bacteriumStreptomyces hygroscopicus.

TABLE G Corn Traits That Can Be Combined With the Insecticidal ProteinsZea mays L. Maize Event Company Description 176 Syngenta Seeds, Inc.Insect-resistant maize produced by inserting the Cry1Ab gene fromBacillus thuringiensis subsp. kurstaki. The genetic modification affordsresistance to attack by the European corn borer (ECB). 3751 IR PioneerHi-Bred Selection of somaclonal variants by 676, 678, 680 InternationalInc. culture of embryos on Pioneer Hi-Bred imidazolinone containingmedia. International Inc. Male-sterile and glufosinate ammoniumherbicide tolerant maize produced by inserting genes encoding DNAadenine methylase and phosphinothricin acetyltransferase (PAT) fromEscherichia coli and Streptomyces viridochromogenes, respectively. B16(DLL25) Dekalb Genetics Glufosinate ammonium herbicide Corporationtolerant maize produced by inserting the gene encoding phosphinothricinacetyltransferase (PAT) from Streptomyces hygroscopicus. BT11 (X4334CBR,Syngenta Seeds, Inc. Insect-resistant and herbicide X4734CBR) tolerantmaize produced by inserting the Cry1Ab gene from Bacillus thuringiensissubsp. kurstaki, and the phosphinothricin N- acetyltransferase (PAT)encoding gene from S. viridochromogenes. BT11 × GA21 Syngenta Seeds,Inc. Stacked insect resistant and herbicide tolerant maize produced byconventional cross breeding of parental lines BT11 (OECD uniqueidentifier: SYN-BTO11-1) and GA21 (OECD unique identifier: MON-OOO21-9).BT11 × MIR162 × Syngenta Seeds, Inc. Resistance to Coleopteran pests,MIR604 × GA21 particularly corn rootworm pests (Diabrotica spp.) andseveral Lepidopteran pests of corn, including European corn borer (ECB,Ostrinia nubilalils), corn earworm (CEW, Helicoverpa zea), fall armyworm (FAW, Spodoptera frupperda), and black cutworm (BCW, Agrotisipsilon); tolerance to glyphosate and glufosinate- ammonium containingherbicides. BT11 × MIR162 Syngenta Seeds, Inc. Stacked insect resistantand herbicide tolerant maize produced by conventional cross breeding ofparental lines BT11 (OECD unique identifier: SYN-BTO11-1) and MIR162(OECD unique identifier: SYN-1R162-4). Resistance to the European CornBorer and tolerance to the herbicide glufosinate ammonium (Liberty) isderived from BT11, which contains the Cry1Ab gene from Bacillusthuringiensis subsp. kurstaki, and the phosphinothricin N-acetyltransferase (PAT) encoding gene from S. viridochromogenes.Resistance to other Lepidopteran pests, including H. zea, S. frupperda,A. ipsilon, and S. albicosta, is derived from MIR162, which contains thevip3Aa gene from Bacillus thuringiensis strain AB88. BT11 × MIR162 ×Syngenta Seeds, Inc. Bacillus thuringiensis Cry1Ab delta- MIR604endotoxin protein and the genetic material necessary for its production(via elements of vector pZO1502) in Event Bt11 corn (OECD UniqueIdentifier: SYNBTO11-1) × Bacillus thuringiensis Vip3Aa20 insecticidalprotein and the genetic material necessary for its production (viaelements of vector pNOV1300) in Event MIR162 maize (OECD UniqueIdentifier: SYN-IR162-4) × modified Cry3A protein and the geneticmaterial necessary for its production (via elements of vector pZM26) inEvent MIR604 corn (OECD Unique Identifier: SYN- 1R604-5). CBH-351Aventis CropScience Insect-resistant and glufosinate ammonium herbicidetolerant maize developed by inserting genes encoding Cry9C protein fromBacillus thuringiensis subsp tolworthi and phosphinothricinacetyltransferase (PAT) from Streptomyces hygroscopicus. DAS-06275-8 DOWAgroSciences LLC Lepidopteran insect resistant and glufosinate ammoniumherbicide- tolerant maize variety produced by inserting the Cry1F genefrom Bacillus thuringiensis var aizawai and the phosphinothricinacetyltransferase (PAT) from Streptomyces hygroscopicus. BT11 × MIR604Syngenta Seeds, Inc. Stacked insect resistant and herbicide tolerantmaize produced by conventional cross breeding of parental lines BT11(OECD unique identifier: SYN-BTO11-1) and MIR604 (OECD uniqueidentifier: SYN-1R6O5-5). Resistance to the European Corn Borer andtolerance to the herbicide glufosinate ammonium (Liberty) is derivedfrom BT11, which contains the Cry1Ab gene from Bacillus thuringiensissubsp. kurstaki, and the phosphinothricin N- acetyltransferase (PAT)encoding gene from S. viridochromogenes. Corn rootworm-resistance isderived from MIR604 which contains the mCry3A gene from Bacillusthuringiensis. BT11 × MIR604 × GA21 Syngenta Seeds, Inc. Stacked insectresistant and herbicide tolerant maize produced by conventional crossbreeding of parental lines BT11 (OECD unique identifier: SYN-BTO11-1),MIR604 (OECD unique identifier: SYN- 1R6O5-5) and GA21 (OECD uniqueidentifier: MON-OOO21-9). Resistance to the European Corn Borer andtolerance to the herbicide glufosinate ammonium (Liberty) is derivedfrom BT11, which contains the Cry1Ab gene from Bacillus thuringiensissubsp. kurstaki, and the phosphinothricin N- acetyltransferase (PAT)encoding gene from S. viridochromogenes. Corn rootworm-resistance isderived from MIR604 which contains the mCry3A gene from Bacillusthuringiensis. Tolerance to glyphosate herbicide is derived from GA21which contains a a modified EPSPS gene from maize. DAS-59122-7 DOWAgroSciences LLC Corn rootworm-resistant maize and Pioneer Hi-Bredproduced by inserting the Cry34Ab1 International Inc. and Cry35Ab1 genesfrom Bacillus thuringiensis strain PS149B1. The PAT encoding gene fromStreptomyces viridochromogenes was introduced as a selectable marker.DAS-59122-7 × DOW AgroSciences LLC Stacked insect resistant and TC1507 ×NK603 and Pioneer Hi-Bred herbicide tolerant maize producedInternational Inc. by conventional cross breeding of parental linesDAS-59122-7 (OECD unique identifier: DAS-59122-7) and TC1507 (OECDunique identifier: DAS-01507-1) with NK603 (OECD unique identifier:MON-00603-6). Corn rootworm- resistance is derived from DAS- 59122-7which contains the Cry34Abl and Cry35Abl genes from Bacillusthuringiensis strain P5149B1. Lepidopteran resistance and tolerance toglufosinate ammonium herbicide is derived from TC1507. Tolerance toglyphosate herbicide is derived from NK603. DBT418 Dekalb GeneticsInsect-resistant and glufosinate Corporation ammonium herbicide tolerantmaize developed by inserting genes encoding Cry1AC protein from Bacillusthuringiensis subsp kurstaki and phosphinothricin acetyltransferase(PAT) from Streptomyces hygroscopicus. MIR604 × GA21 Syngenta Seeds,Inc. Stacked insect resistant and herbicide tolerant maize produced byconventional cross breeding of parental lines MIR604 (OECD uniqueidentifier: SYN-1R605-5) and GA21 (OECD unique identifier: MON-00021-9).Com rootworm- resi stance is derived from MIR604 which contains themCry3A gene from Bacillus thuringiensis. Tolerance to glyphosateherbicide is derived from GA21. MON80100 Monsanto CompanyInsect-resistant maize produced by inserting the Cry1Ab gene fromBacillus thuringiensis subsp. kurstaki. The genetic modification affordsresistance to attack by the European corn borer (ECB). MON802 MonsantoCompany Insect-resistant and glyphosate herbicide tolerant maizeproduced by inserting the genes encoding the Cry1Ab protein fromBacillus thuringiensis and the 5- enolpyruvylshikimate-3-phosphatesynthase (EPSPS) from A. tumefaciens strain CP4. MON809 Pioneer Hi-BredResistance to European corn borer International Inc. (Ostrinianubilalis) by introduction of a synthetic Cry1Ab gene. Glyphosateresistance via introduction of the bacterial version of a plant enzyme,5-enolpynivyl shikimate-3-phosphate synthase (EPSPS). MON810 MonsantoCompany Insect-resistant maize produced by inserting a truncated form ofthe Cry1Ab gene from Bacillus thuringiensis subsp. kurstaki HD-1. Thegenetic modification affords resistance to attack by the European cornborer (ECB). MON810 × LY038 Monsanto Company Stacked insect resistantand enhanced lysine content maize derived from conventionalcrossbreeding of the parental lines MON810 (OECD identifier: MON-OO81O-6) and LY038 (OECD identifier: REN-OOO38-3). MON810 × MON88017Monsanto Company Stacked insect resistant and glyphosate tolerant maizederived from conventional cross-breeding of the parental lines MON810(OECD identifier: MON-OO81O-6) and MON88017 (OECD identifier:MON-88017-3). European corn borer (ECB) resistance is derived from atruncated form of the Cry1Ab gene from Bacillus thuringiensis subsp.kurstaki HD-1 present in MON810. Corn rootworm resistance is derivedfrom the Cry3Bbl gene from Bacillus thuringiensis subspecieskumamotoensis strain EG4691 present in MON88017. Glyphosate tolerance isderived from a 5- enolpyruvylshikimate-3-phosphate synthase (EPSPS)encoding gene from Agrobacterium tumefaciens strain CP4 present inMON88017. MON832 Monsanto Company Introduction, by particle bombardment,of glyphosate oxidase (GOX) and a modified 5- enolpyruvylshikimate-3-phosphate synthase (EPSPS), an enzyme involved in theshikimate biochemical pathway for the production of the aromatic aminoacids. MON863 Monsanto Company Corn rootworm resistant maize produced byinserting the Cry3Bbl gene from Bacillus thuringiensis subsp.kumamotoensis. MON863 × MON810 Monsanto Company Stacked insect resistantcorn hybrid derived from conventional cross- breeding of the parentallines MON863 (OECD identifier: MON- 00863-5) and MON810 (OECDidentifier: MON-00810-6) MON863 × MON810 × Monsanto Company Stackedinsect resistant and Monsanto NK603 herbicide tolerant corn hybridderived from conventional crossbreeding of the stacked hybridMON-00863-5 × MON-00810-6 and NK603 (OECD identifier: MON- 00603-6).MON863 × NK603 Monsanto Company Stacked insect resistant and herbicidetolerant corn hybrid derived from conventional crossbreeding of theparental lines MON863 (OECD identifier: MON- OO863-5) and NK603 (OECDidentifier: MON-OO6O3-6). MON87460 Monsanto Company MON 87460 wasdeveloped to provide reduced yield loss under water-limited conditionscompared to conventional maize. Efficacy in MON 87460 is derived byexpression of the inserted Bacillus subtilis cold shock protein B(CspB). MON88017 Monsanto Company Corn rootworm-resistant maize producedby inserting the Cry3Bbl gene from Bacillus thuringiensis subspecieskumamotoensis strain EG4691. Glyphosate tolerance derived by inserting a5- enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene fromAgrobacterium tumefaciens strain CP4. MON89034 Monsanto Company Maizeevent expressing two different insecticidal proteins from Bacillusthuringiensis providing resistance to number of Lepidopteran pests.MON89034 × Monsanto Company Stacked insect resistant and MON88017glyphosate tolerant maize derived from conventional cross-breeding ofthe parental lines MON89034 (OECD identifier: MON-89O34-3) and MON88017(OECD identifier: MON-88O17-3). Resistance to Lepidopteran insects isderived from two Cry genes present in MON89043. Corn rootworm resistanceis derived from a single Cry genes and glyphosate tolerance is derivedfrom the 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) encodinggene from Agrobacterium tumefaciens present in MON88017. MON89034 ×NK603 Monsanto Company Stacked insect resistant and herbicide tolerantmaize produced by conventional cross breeding of parental lines MON89034(OECD identifier: MON-89034-3) with NK603 (OECD unique identifier:MON-00603-6). Resistance to Lepidopteran insects is derived from two Crygenes present in MON89043. Tolerance to glyphosate herbicide is derivedfrom NK603. NK603 × MON810 Monsanto Company Stacked insect resistant andherbicide tolerant corn hybrid derived from conventional crossbreedingof the parental lines NK603 (OECD identifier: MON- 00603-6) and MON810(OECD identifier: MON-00810-6). MON89034 × TC1507 × Monsanto Company andStacked insect resistant and MON88017 × DAS- Mycogen Seeds c/o Dowherbicide tolerant maize produced 59122-7 AgroSciences LLC byconventional cross breeding of parental lines: MON89034, TC1507,MON88017, and DAS-59 122. Resistance to the above-ground andbelow-ground insect pests and tolerance to glyphosate andglufosinate-ammonium containing herbicides. M53 Bayer CropScience Malesterility caused by expression (Aventis of the barnase ribonuclease geneCropScience(AgrEvo)) from Bacillus amyloliquefaciens; PPT resistance wasvia PPT- acetyltransferase (PAT). M56 Bayer CropScience Male sterilitycaused by expression (Aventis of the barnase ribonuclease geneCropScience(AgrEvo) from Bacillus amyloliquefaciens; PPT resistance wasvia PPT- acetyltransferase (PAT). NK603 Monsanto Company Introduction,by particle bombardment, of a modified 5- enolpyruvylshikimate-3-phosphate synthase (EPSPS), an enzyme involved in theshikimate biochemical pathway for the production of the aromatic aminoacids. NK603 × T25 Monsanto Company Stacked glufosinate ammonium andglyphosate herbicide tolerant maize hybrid derived from conventionalcross-breeding of the parental lines NK603 (OECD identifier: MON-00603-6) and T25 (OECD identifier: ACS-ZM003-2). T25 × MON810 BayerCropScience Stacked insect resistant and (Aventis herbicide tolerantcorn hybrid CropScience(AgrEvo)) derived from conventional crossbreedingof the parental lines T25 (OECD identifier: ACS- ZMOO3-2) and MON810(OECD identifier: MON-OO81O-6). TC1507 Mycogen (c/o Dow Insect-resistantand glufosinate AgroSciences); Pioneer ammonium herbicide tolerant maize(c/o DuPont) produced by inserting the Cry1F gene from Bacillusthuringiensis var. aizawai and the phosphinothricin N-acetyltransferaseencoding gene from Streptomyces viridochromogenes. TC1507 × NK603 DOWAgroSciences LLC Stacked insect resistant and herbicide tolerant cornhybrid derived from conventional crossbreeding of the parental lines1507 (OECD identifier: DAS- O15O7-1) and NK603 (OECD identifier:MON-OO6O3-6). TC1507 × DAS-59122-7 DOW AgroSciences LLC Stacked insectresistant and and Pioneer Hi-Bred herbicide tolerant maize producedInternational Inc. by conventional cross breeding of parental linesTC1507 (OECD unique identifier: DAS-O15O7-1) with DAS-59122-7 (OECDunique identifier: DAS-59122-7). Resistance to Lepidopteran insects isderived from TC1507 due the presence of the Cry1F gene from Bacillusthuringiensis var. aizawai. Corn rootworm-resistance is derived fromDAS-59122-7 which contains the Cry34Ab1 and Cry35Ab1 genes from Bacillusthuringiensis strain P5149B1. Tolerance to glufosinate ammoniumherbicide is derived from TC1507 from the phosphinothricinN-acetyltransferase encoding gene from Streptomyces viridochromogenes.

Utilization of Microbes to Express the Insecticidal Proteins

Microorganism hosts that are known to occupy the “phytosphere”(phylloplane, phyllosphere, rhizosphere, and/or rhizoplana) of one ormore crops of interest may be selected. These microorganisms areselected so as to be capable of successfully competing in the particularenvironment with the wild-type microorganisms, provide for stablemaintenance, and expression of the gene expressing the pesticidalproteins taught herein, and provide for improved protection of thepesticide from environmental degradation and inactivation.

Such microorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms such as bacteria, e.g., Pseudomonas,Envinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,Rhodopseudomonas, Methylius, Agrobacterium, Acetobacter, Lactobacillus,Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes, fungi,particularly yeast, e.g., Saccharomyces, Cryptococcus, Kluyveromyces,Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interestare such phytosphere bacterial species as Pseudomonas syringae,Pseudomonas jluorescens, Pseudomonas chlororaphis, Serratia marcescens,Acetobacter xylinum, Agrobacteria, Rhodopseudomonas spheroides,Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus,Clavibacter xyli and Azotobacter vinelandii and phytosphere yeastspecies such as Rhodotorula rubra, R. glutinis, R. marina, R.aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii,Saccharomyces rosei, S. pretoriensis, S. cerevzszae, Sporobolomycesroseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans.Of particular interest are the pigmented microorganisms. Host organismsof particular interest include yeast, such as Rhodotorula spp.,Aureobasidium spp., Saccharomyces spp. (such as S. cerevisiae),Sporobolomyces spp., phylloplane organisms such as Pseudomonas spp.(such as P. aeruginosa, P. fluorescens, P. chlororaphis), Erwinia spp.,and Flavobacterium spp., and other such organisms, includingAgrobacterium tumefaciens, E. coli, Bacillus subtilis, Bacillus cereusand the like.

Genes encoding the taught pesticidal proteins can be introduced intomicroorganisms that multiply on plants (epiphytes). Epiphytes can begram positive or gram negative bacteria. Root colonizing bacteria can beisolated from the plant of interest by methods known in the art. Genesencoding the taught pesticidal proteins can be introduced, for example,into the root colonizing or epiphytic bacteria by means of electrotransformation. Genes can be cloned into a shuttle vector, for example,pHT3101 (Lerecius, et al., (1989) FEMS Microbiol. Lett. 60:211-218. Theshuttle vector pHT3101 containing the coding sequence for the particularpolypeptide gene can, for example, be transformed into the bacteria bymeans of electroporation (Lerecius, et al., (1989) FEMS Microbiol. Lett.60:211-218). Expression systems can be designed so that the taughtpesticidal proteins are secreted outside the cytoplasm of gram negativebacteria, such as E. coli, for example.

Pesticidal proteins taught herein may be fermented in a bacterial hostand the resulting bacteria processed and used as a microbial spray inthe same manner that Bt strains have been used as insecticidal sprays.In the case of a pesticidal protein that is secreted from Bacillus, thesecretion signal is removed or mutated using procedures known in theart. Such mutations and/or deletions prevent secretion of the proteininto the growth medium during the fermentation process. The pesticidalproteins are retained within the cell and the cells are then processedto yield the encapsulated proteins. Any suitable microorganism can beused for this purpose. Pseudomonas has been used to express Bt toxins asencapsulated proteins and the resulting cells processed and sprayed asan insecticide (Gaertner, et al., (1993), in: Advanced EngineeredPesticides, ed. Kim).

Alternatively, the taught pesticidal proteins are produced byintroducing a heterologous gene into a cellular host. Expression of theheterologous gene results, directly or indirectly, in the intracellularproduction and maintenance of the pesticide. These cells are thentreated under conditions that prolong the activity of the toxin producedin the cell when the cell is applied to the environment of targetpest(s). The resulting product retains the toxicity of the toxin. Thesenaturally encapsulated proteins may then be formulated in accordancewith conventional techniques for application to the environment hostinga target pest, e.g., soil, water, and foliage of plants. See, forexample EPA 0192319, and the references cited therein.

Pesticidal Compositions

In some embodiments the active ingredients can be applied in the form ofcompositions and can be applied to the crop area or plant to be treated,simultaneously or in succession, with other compounds. These compoundscan be fertilizers, weed killers, Cryoprotectants, surfactants,detergents, pesticidal soaps, dormant oils, polymers, and/or timerelease or biodegradable carrier formulations that permit long termdosing of a target area following a single application of theformulation. They can also be selective herbicides, chemicalinsecticides, virucides, microbicides, amoebicides, pesticides,fungicides, bacteriocides, nematocides, molluscicides or mixtures ofseveral of these preparations, if desired, together with furtheragriculturally acceptable carriers, surfactants or application promotingadjuvants customarily employed in the art of formulation. Suitablecarriers (i.e. agriculturally acceptable carriers) and adjuvants can besolid or liquid and correspond to the substances ordinarily employed informulation technology, e.g. natural or regenerated mineral substances,solvents, dispersants, wetting agents, sticking agents, tackifiers,binders or fertilizers. Likewise the formulations may be prepared intoedible baits or fashioned into pest traps to permit feeding or ingestionby a target pest of the pesticidal formulation.

Methods of applying an active ingredient or an agrochemical compositionthat contains at least one of the taught insecticidal proteins producedby the bacterial strains include leaf application, seed coating, andsoil application. The number of applications and the rate of applicationdepend on the intensity of infestation by the corresponding pest.

The composition may be formulated as a powder, dust, pellet, granule,spray, emulsion, colloid, solution or such like, and may be prepared bysuch conventional means as desiccation, lyophilization, homogenization,extraction, filtration, centrifugation, sedimentation or concentrationof a culture of cells comprising the polypeptide. In all suchcompositions that contain at least one such pesticidal polypeptide, thepolypeptide may be present in a concentration of from about 1% to about99% by weight.

Lepidopterans, Dipterans, Hemipterans, Heteropterans, Nematodes, orColeopterans may be killed or reduced in numbers in a given area by themethods of the disclosure or may be prophylactically applied to anenvironmental area to prevent infestation by a susceptible pest.Preferably the pest ingests or is contacted with, a pesticidallyeffective amount of the disclosed insecticidal protein. A “Pesticidallyeffective amount” refers to an amount of the pesticide that is able tobring about death to at least one pest or to noticeably reduce pestgrowth, feeding, or normal physiological development. This amount willvary depending on such factors as, for example: the specific targetpests to be controlled, the specific environment, location, plant, cropor agricultural site to be treated, the environmental conditions and themethod, rate, concentration, stability, and quantity of application ofthe pesticidally effective protein composition. The formulations mayalso vary with respect to climatic conditions, environmentalconsiderations, and/or frequency of application and/or severity of pestinfestation.

The pesticide compositions described may be made by formulating eitherthe bacterial cell, Crystal and/or spore suspension, or isolated proteincomponent with the desired agriculturally acceptable carrier.

The compositions may be formulated prior to administration in anappropriate means such as lyophilized, freeze dried, desiccated or in anaqueous carrier, medium or suitable diluent, such as saline or otherbuffer. The formulated compositions may be in the form of a dust orgranular material or a suspension in oil (vegetable or mineral) or wateror oil/water emulsions or as a wettable powder or in combination withany other carrier material suitable for agricultural application.Suitable agricultural carriers can be solid or liquid and are well knownin the art. The term “agriculturally acceptable carrier” covers alladjuvants, inert components, dispersants, surfactants, stickers,tackifiers, binders, etc. that are ordinarily used in pesticideformulation technology; these are well known to those skilled inpesticide formulation. The formulations may be mixed with one or moresolid or liquid adjuvants and prepared by various means, e.g., byhomogeneously mixing, blending and/or grinding the pesticidalcomposition with suitable adjuvants using conventional formulationtechniques. Suitable formulations and application methods are describedin U.S. Pat. No. 6,468,523, herein incorporated by reference. The plantscan also be treated with one or more chemical compositions, includingone or more herbicide, insecticides or fungicides.

Exemplary chemical compositions include: Fruits/Vegetables Herbicides:Atrazine, Bromacil, Diuron, Glyphosate, Linuron, Metribuzin, Simazine,Trifluralin, Fluazifop, Glufosinate, Halo sulfuron Gowan, Paraquat,Propyzamide, Sethoxydim, Butafenacil, Halosulfuron, Indaziflam;Fruits/Vegetables Insecticides: Aldicarb, Bacillus thuringiensis,Carbaryl, Carbofuran, Chlorpyrifos, Cypermethrin, Deltamethrin,Diazinon, Malathion, Abamectin, Cyfluthrin/betacyfluthrin,Esfenvalerate, Lambda-cyhalothrin, Acequinocyl, Bifenazate,Methoxyfenozide, Novaluron, Chromafenozide, Thiacloprid, Dinotefuran,FluaCrypyrim, Tolfenpyrad, Clothianidin, Spirodiclofen,Gamma-cyhalothrin, Spiromesifen, Spinosad, Rynaxypyr, Cyazypyr,Spinetoram, Triflumuron, Spirotetramat, Imidacloprid, Flubendiamide,Thiodicarb, Metaflumizone, Sulfoxaflor, Cyflumetofen, Cyanopyrafen,Imidacloprid, Clothianidin, Thiamethoxam, Spinetoram, Thiodicarb,Flonicamid, Methiocarb, Emamectin benzoate, Indoxacarb, Forthiazate,Fenamiphos, Cadusaphos, Pyriproxifen, Fenbutatin oxide, Hexthiazox,Methomyl, 4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on; Fruits Vegetables Fungicides:Carbendazim, Chlorothalonil, EBDCs, Sulphur, Thiophanate-methyl,Azoxystrobin, Cymoxanil, Fluazinam, Fosetyl, Iprodione, Kresoxim-methyl,Metalaxyl/mefenoxam, Trifloxystrobin, Ethaboxam, Iprovalicarb,Trifloxystrobin, Fenhexamid, Oxpoconazole fumarate, Cyazofamid,Fenamidone, Zoxamide, Picoxystrobin, Pyraclostrobin, Cyflufenamid,Boscalid; Cereals Herbicides: Isoproturon, Bromoxynil, loxynil,Phenoxies, Chlorsulfuron, Clodinafop, Diclofop, Diflufenican,Fenoxaprop, Florasulam, Fluoroxypyr, Metsulfuron, Triasulfuron,Flucarbazone, lodosulfuron, Propoxycarbazone, Picolinafen, Mesosulfuron,Beflubutamid, Pinoxaden, Amidosulfuron, Thifensulfuron Methyl,Tribenuron, Flupyrsulfuron, Sulfosulfuron, Pyrasulfotole, Pyroxsulam,Flufenacet, Tralkoxydim, Pyroxasulfon; Cereals Fungicides: Carbendazim,Chlorothalonil, Azoxystrobin, Cyproconazole, Cyprodinil, Fenpropimorph,Epoxiconazole, Kresoxim-methyl, Quinoxyfen, Tebuconazole,Trifloxystrobin, Simeconazole, Picoxystrobin, Pyraclostrobin,Dimoxystrobin, Prothioconazole, Fluoxastrobin; Cereals Insecticides:Dimethoate, Lambda-cyhalothrin, Deltamethrin, alpha-Cypermethrin,β-cyfluthrin, Bifenthrin, Imidacloprid, Clothianidin, Thiamethoxam,Thiacloprid, Acetamiprid, Dinetofuran, Clorphyriphos, Metamidophos,Oxidemethon methyl, Pirimicarb, Methiocarb; Maize Herbicides: Atrazine,Alachlor, Bromoxynil, Acetochlor, Dicamba, Clopyralid, S-Dimethenamid,Glufosinate, Glyphosate, Isoxaflutole, S-Metolachlor, Mesotrione,Nicosulfuron, Primisulfuron, Rimsulfuron, Sulcotrione, Foramsulfuron,Topramezone, Tembotrione, Saflufenacil, Thiencarbazone, Flufenacet,Pyroxasulfon; Maize Insecticides: Carbofuran, Chlorpyrifos, Bifenthrin,Fipronil, Imidacloprid, Lambda-Cyhalothrin, Tefluthrin, Terbufos,Thiamethoxam, Clothianidin, Spiromesifen, Flubendiamide, Triflumuron,Rynaxypyr, Deltamethrin, Thiodicarb, β-Cyfluthrin, Cypermethrin,Bifenthrin, Lufenuron, Triflumoron, Tefluthrin, Tebupirimphos,Ethiprole, Cyazypyr, Thiacloprid, Acetamiprid, Dinetofuran, Avermectin,Methiocarb, Spirodiclofen, Spirotetramat; Maize Fungicides: Fenitropan,Thiram, Prothioconazole, Tebuconazole, Trifloxystrobin; Rice Herbicides:Butachlor, Propanil, Azimsulfuron, Bensulfuron, Cyhalofop, Daimuron,Fentrazamide, Imazosulfuron, Mefenacet, Oxaziclomefone, Pyrazosulfuron,Pyributicarb, Quinclorac, Thiobencarb, Indanofan, Flufenacet,Fentrazamide, Halosulfuron, Oxaziclomefone, Benzobicyclon, Pyriftalid,Penoxsulam, Bispyribac, Oxadiargyl, Ethoxysulfuron, Pretilachlor,Mesotrione, Tefuryltrione, Oxadiazone, Fenoxaprop, Pyrimisulfan; RiceInsecticides: Diazinon, Fenitrothion, Fenobucarb, Monocrotophos,Benfuracarb, Buprofezin, Dinotefuran, Fipronil, Imidacloprid,Isoprocarb, Thiacloprid, Chromafenozide, Thiacloprid, Dinotefuran,Clothianidin, Ethiprole, Flubendiamide, Rynaxypyr, Deltamethrin,Acetamiprid, Thiamethoxam, Cyazypyr, Spinosad, Spinetoram,Emamectin-Benzoate, Cypermethrin, Chlorpyriphos, Cartap, Methamidophos,Etofenprox, Triazophos,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Carbofuran, Benfuracarb; Rice Fungicides: Thiophanate-methyl,Azoxystrobin, Carpropamid, Edifenphos, Ferimzone, Iprobenfos,Isoprothiolane, Pencycuron, Probenazole, Pyroquilon, Tricyclazole,Trifloxystrobin, Diclocymet, Fenoxanil, Simeconazole, Tiadinil; CottonHerbicides: Diuron, Fluometuron, MSMA, Oxyfluorfen, Prometryn,Trifluralin Carfentrazone, Clethodim, Fluazifop-butyl, Glyphosate,Norflurazon, Pendimethalin, Pyrithiobac-sodium, Trifloxysulfuron,Tepraloxydim, Glufosinate, Flumioxazin, Thidiazuron; CottonInsecticides: Acephate, Aldicarb, Chlorpyrifos, Cypermethrin,Deltamethrin, Malathion, Monocrotophos, Abamectin, Acetamiprid,Emamectin Benzoate, Imidacloprid, Indoxacarb, Lambda-Cyhalothrin,Spinosad, Thiodicarb, Gamma-Cyhalothrin, Spiromesifen, Pyridalyl,Flonicamid, Flubendiamide, Triflumuron, Rynaxypyr, Beta-Cyfluthrin,Spirotetramat, Clothianidin, Thiamethoxam, Thiacloprid, Dinetofuran,Flubendiamide, Cyazypyr, Spinosad, Spinetoram, gamma Cyhalothrin,4-[[(6-Chlorpyridin-3-yl) methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Thiodicarb, Avermectin, Flonicamid, Pyridalyl, Spiromesifen,Sulfoxaflor, Profenophos, Thriazophos, Endosulfan; Cotton Fungicides:Etridiazole, Metalaxyl, Quintozene; Soybean Herbicides: Alachlor,Bentazone, Trifluralin, Chlorimuron-Ethyl, Cloransulam-Methyl,Fenoxaprop, Fomesafen, Fluazifop, Glyphosate, Imazamox, Imazaquin,Imazethapyr, (S-)Metolachlor, Metribuzin, Pendimethalin, Tepraloxydim,Glufosinate; Soybean Insecticides: Lambda-cyhalothrin, Methomyl,Parathion, Thiocarb, Imidacloprid, Clothianidin, Thiamethoxam,Thiacloprid, Acetamiprid, Dinetofuran, Flubendiamide, Rynaxypyr,Cyazypyr, Spinosad, Spinetoram, Emamectin-Benzoate, Fipronil, Ethiprole,Deltamethrin, β-Cyfluthrin, gamma and lambda Cyhalothrin,4-[[(6-Chlorpyridin-3-yl)methyl] (2,2-difluorethyl)amino]furan-2(5H)-on,Spirotetramat, Spinodiclofen, Triflumuron, Flonicamid, Thiodicarb,beta-Cyfluthrin; Soybean Fungicides: Azoxystrobin, Cyproconazole,Epoxiconazole, Flutriafol, Pyraclostrobin, Tebuconazole,Trifloxystrobin, Prothioconazole, Tetraconazole; Sugarbeet Herbicides:Chloridazon, Desmedipham, Ethofumesate, Phenmedipham, Triallate,Clopyralid, Fluazifop, Lenacil, Metamitron, Quinmerac, Cycloxydim,Triflusulfuron, Tepraloxydim, Quizalofop; Sugarbeet Insecticides:Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid,Dinetofuran, Deltamethrin, β-Cyfluthrin, gamma/lambda Cyhalothrin,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Tefluthrin, Rynaxypyr, Cyaxypyr, Fipronil, Carbofuran; CanolaHerbicides: Clopyralid, Diclofop, Fluazifop, Glufosinate, Glyphosate,Metazachlor, Trifluralin Ethametsulfuron, Quinmerac, Quizalofop,Clethodim, Tepraloxydim; Canola Fungicides: Azoxystrobin, Carbendazim,Fludioxonil, Iprodione, Prochloraz, Vinclozolin; Canola Insecticides:Carbofuran organophosphates, Pyrethroids, Thiacloprid, Deltamethrin,Imidacloprid, Clothianidin, Thiamethoxam, Acetamiprid, Dinetofuran,β-Cyfluthrin, gamma and lambda Cyhalothrin, tau-Fluvaleriate, Ethiprole,Spinosad, Spinetoram, Flubendiamide, Rynaxypyr, Cyazypyr,4-[[(6-Chlorpyridin-3-yl)methyl] (2,2-difluorethyl)amino]furan-2(5H)-on.

Pests

“Pest” includes but is not limited to, insects, fungi, bacteria,nematodes, mites, ticks and the like. Insect pests include insectsselected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera,Mallophaga, Homoptera, Hemiptera Orthroptera, Thysanoptera, Dermaptera,Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularlyLepidoptera and Coleoptera.

Those skilled in the art will recognize that not all compounds areequally effective against all pests. Compounds of the embodimentsdisplay activity against insect pests, which may include economicallyimportant agronomic, forest, greenhouse, nursery ornamentals, food andfiber, public and animal health, domestic and commercial structure,household and stored product pests.

Larvae of the order Lepidoptera include, but are not limited to,armyworms, cutworms, loopers and heliothines in the family NoctuidaeSpodoptera frugiperda J E Smith (fall armyworm); S. exigua Hubner (beetarmyworm); S. litura Fabricius (tobacco cutworm, cluster caterpillar);Mamestra configurata Walker (bertha armyworm); M. brassicae Linnaeus(cabbage moth); Agrotis ipsilon Hufnagel (black cutworm); A. orthogoniaMorrison (western cutworm); A. subterranea Fabricius (granulatecutworm); Alabama argillacea Hubner (cotton leaf worm); Trichoplusia niHubner (cabbage looper); Pseudoplusia includens Walker (soybean looper);Anticarsia gemmatalis Hubner (velvet bean caterpillar); Hypena scabraFabricius (green clover worm); Heliothis virescens Fabricius (tobaccobudworm); Pseudaletia unipuncta Haworth (armyworm); Athetis mindaraBarnes and Mcdunnough (rough skinned cutworm); Euxoa messoria Harris(darksided cutworm); Earias insulana Boisduval (spiny bollworm); E.vittella Fabricius (spotted bollworm); Helicoverpa armigera Hubner(American bollworm); H. zea Boddie (corn earworm or cotton bollworm);Melanchra picta Harris (zebra caterpillar); Egira (Xylomyges) curialisGrote (citrus cutworm); borers, case bearers, webworms, coneworms, andskeletonizers from the family Pyralidae Ostrinia nubilalis Hubner(European corn borer); Amyelois transitella Walker (naval orangeworm);Anagasta kuehniella Zeller (Mediterranean flour moth); Cadra cautellaWalker (almond moth); Chilo suppressalis Walker (rice stem borer); C.partellus, (sorghum borer); Corcyra cephalonica Stainton (rice moth);Crambus caliginosellus Clemens (corn root webworm); C. teterrellusZincken (bluegrass webworm); Cnaphalocrocis medinalis Guenee (rice leafroller); Desmia funeralis Hubner (grape leaffolder); Diaphania hyalinataLinnaeus (melon worm); D. nitidalis Stoll (pickleworm); Diatraeagrandiosella Dyar (southwestern corn borer), D. saccharalis Fabricius(surgarcane borer); Eoreuma loftini Dyar (Mexican rice borer); Ephestiaelutella Hubner (tobacco (cacao) moth); Galleria mellonella Linnaeus(greater wax moth); Herpetogramma licarsisalis Walker (sod webworm);Homoeosoma electellum Hulst (sunflower moth); Elasmopalpus lignosellusZeller (lesser cornstalk borer); Achroia grisella Fabricius (lesser waxmoth); Loxostege sticticalis Linnaeus (beet webworm); Orthaga thyrisalisWalker (tea tree web moth); Maruca testulalis Geyer (bean pod borer);Plodia interpunctella Hubner (Indian meal moth); Scirpophaga incertulasWalker (yellow stem borer); Udea rubigalis Guenee (celery leaftier); andleafrollers, budworms, seed worms and fruit worms in the familyTortricidae Acleris gloverana Walsingham (Western blackheaded budworm);A. variana Fernald (Eastern blackheaded budworm); Archips argyrospilaWalker (fruit tree leaf roller); A. rosana Linnaeus (European leafroller); and other Archips species, Adoxophyes orana Fischer vonRosslerstamm (summer fruit tortrix moth); Cochylis hospes Walsingham(banded sunflower moth); Cydia latiferreana Walsingham (filbertworm); C.pomonella Linnaeus (colding moth); Platynota flavedana Clemens(variegated leafroller); P. stultana Walsingham (omnivorous leafroller);Lobesia botrana Denis & Schiffermuller (European grape vine moth);Spilonota ocellana Denis & Schiffermuller (eyespotted bud moth);Endopiza viteana Clemens (grape berry moth); Eupoecilia ambiguellaHubner (vine moth); Bonagota salubricola Meyrick (Brazilian appleleafroller); Grapholita molesta Busck (oriental fruit moth); Suleimahelianthana Riley (sunflower bud moth); Argyrotaenia spp.; Choristoneuraspp.

Selected other agronomic pests in the order Lepidoptera include, but arenot limited to, Alsophila pometaria Harris (fall cankerworm); Anarsialineatella Zeller (peach twig borer); Anisota senatoria J. E. Smith(orange striped oakworm); Antheraea pernyi Guerin-Meneville (Chinese OakTussah Moth); Bombyx mori Linnaeus (Silkworm); Bucculatrix thurberiellaBusck (cotton leaf perforator); Colias eurytheme Boisduval (alfalfacaterpillar); Datana integerrima Grote & Robinson (walnut caterpillar);Dendrolimus sibiricus Tschetwerikov (Siberian silk moth), Ennomossubsignaria Hubner (elm spanworm); Erannis tiliaria Harris (lindenlooper); Euproctis chrysorrhoea Linnaeus (browntail moth); Harrisinaamericana Guerin-Meneville (grapeleaf skeletonizer); Hemileuca oliviaeCockrell (range caterpillar); Hyphantria cunea Drury (fall webworm);Keiferia lycopersicella Walsingham (tomato pinworm); Lambdinafiscellaria fiscellaria Hulst (Eastern hemlock looper); L. fiscellarialugubrosa Hulst (Western hemlock looper); Leucoma salicis Linnaeus(satin moth); Lymantria dispar Linnaeus (gypsy moth); Manducaquinquemaculata Haworth (five spotted hawk moth, tomato hornworm); M.sexta Haworth (tomato homworm, tobacco hornworm); Operophtera brumataLinnaeus (winter moth); Paleacrita vernata Peck (spring cankerworm);Papilio cresphontes Cramer (giant swallowtail orange dog); Phryganidiacalifornica Packard (California oakworm); Phyllocnistis citrellaStainton (citrus leafminer); Phyllonorycter blancardella Fabricius(spotted tentiform leafminer); Pieris brassicae Linnaeus (large whitebutterfly); P. rapae Linnaeus (small white butterfly); P. napi Linnaeus(green veined white butterfly); Platyptilia carduidactyla Riley(artichoke plume moth); Plutella xylostella Linnaeus (diamondback moth);Pectinophora gossypiella Saunders (pink bollworm); Pontia protodiceBoisduval and Leconte (Southern cabbageworm); Sabulodes aegrotata Guenee(onmivorous looper); Schizura concinna J. E. Smith (red humpedcaterpillar); Sitotroga cerealella Olivier (Angoumois grain moth);Thaumetopoea pityocampa Schiffermuller (pine processionary caterpillar);Tineola bisselliella Hummel (webbing clothes moth); Tuta absolutaMeyrick (tomato leafminer); Yponomeuta padella Linnaeus (ermine moth);Heliothis subflexa Guenee; Malacosoma spp. and Orgyia spp.

Of interest are larvae and adults of the order Coleoptera includingweevils from the families Anthribidae, Bruchidae and Curculionidae(including, but not limited to: Anthonomus grandis Boheman (bollweevil); Lissorhoptrus oryzophilus Kuschel (rice water weevil);Sitophilus granarius Linnaeus (granary weevil); S. oryzae Linnaeus (riceweevil); Hypera punctata Fabricius (clover leaf weevil);Cylindrocopturus adspersus LeConte (sunflower stem weevil); Smicronyxfulvus LeConte (red sunflower seed weevil); S. sordidus LeConte (graysunflower seed weevil); Sphenophorus maidis Chittenden (maize billbug));flea beetles, cucumber beetles, rootworms, leaf beetles, potato beetlesand leafminers in the family Chrysomelidae (including, but not limitedto: Leptinotarsa decemlineata Say (Colorado potato beetle); Diabroticavirgifera virgifera LeConte (western corn rootworm); D. barberi Smithand Lawrence (northern corn rootworm); D. undecimpunctata howardi Barber(southern corn rootworm); Chaetocnema pulicaria Melsheimer (corn fleabeetle); Phyllotreta cruciferae Goeze (Crucifer flea beetle);Phyllotreta striolata (stripped flea beetle); Colaspis brunnea Fabricius(grape colaspis); Oulema melanopus Linnaeus (cereal leaf beetle);Zygogramma exclamationis Fabricius (sunflower beetle)); beetles from thefamily Coccinellidae (including, but not limited to: Epilachnavarivestis Mulsant (Mexican bean beetle)); chafers and other beetlesfrom the family Scarabaeidae (including, but not limited to: Popilliajaponica Newman (Japanese beetle); Cyclocephala borealis Arrow (northernmasked chafer, white grub); C. immaculata Olivier (southern maskedchafer, white grub); Rhizotrogus majalis Razoumowsky (European chafer);Phyllophaga crinita Burmeister (white grub); Ligyrus gibbosus De Geer(carrot beetle)); carpet beetles from the family Dermestidae; wirewormsfrom the family Elateridae, Eleodes spp., Melanotus spp.; Conoderusspp.; Limonius spp.; Agriotes spp.; Ctenicera spp.; Aeolus spp.; barkbeetles from the family Scolytidae and beetles from the familyTenebrionidae.

Adults and immatures of the order Diptera are of interest, includingleafminers Agromyza parvicornis Loew (corn blotch leafminer); midges(including, but not limited to: Contarinia sorghicola Coquillett(sorghum midge); Mayetiola destructor Say (Hessian fly); Sitodiplosismosellana Gehin (wheat midge); Neolasioptera murtfeldtiana Felt,(sunflower seed midge)); fruit flies (Tephritidae), Oscinella fritLinnaeus (fruit flies); maggots (including, but not limited to: Deliaplatura Meigen (seedcorn maggot); D. coarctata Fallen (wheat bulb fly)and other Delia spp., Meromyza americana Fitch (wheat stem maggot);Musca domestica Linnaeus (house flies); Fannia canicularis Linnaeus, F.femoralis Stein (lesser house flies); Stomoxys calcitrans Linnaeus(stable flies)); face flies, horn flies, blow flies, Chrysomya spp.;Phormia spp. and other muscoid fly pests, horse flies Tabanus spp.; botflies Gastrophilus spp.; Oestrus spp.; cattle grubs Hypoderma spp.; deerflies Chrysops spp.; Melophagus ovinus Linnaeus (keds) and otherBrachycera, mosquitoes Aedes spp.; Anopheles spp.; Culex spp.; blackflies Prosimulium spp.; Simulium spp.; biting midges, sand flies,sciarids, and other Nematocera.

Included as insects of interest are adults and nymphs of the ordersHemiptera and Homoptera such as, but not limited to, adelgids from thefamily Adelgidae, plant bugs from the family Miridae, cicadas from thefamily Cicadidae, leafhoppers, Empoasca spp.; from the familyCicadellidae, planthoppers from the families Cixiidae, Flatidae,Fulgoroidea, Issidae and Delphacidae, treehoppers from the familyMembracidae, psyllids from the family Psyllidae, whiteflies from thefamily Aleyrodidae, aphids from the family Aphididae, phylloxera fromthe family Phylloxeridae, mealybugs from the family Pseudococcidae,scales from the families Asterolecanidae, Coccidae, Dactylopiidae,Diaspididae, Eriococcidae Ortheziidae, Phoenicococcidae andMargarodidae, lace bugs from the family Tingidae, stink bugs from thefamily Pentatomidae, cinch bugs, Blissus spp.; and other seed bugs fromthe family Lygaeidae, spittlebugs from the family Cercopidae squash bugsfrom the family Coreidae and red bugs and cotton stainers from thefamily Pyrrhocoridae.

Agronomically important members from the order Homoptera furtherinclude, but are not limited to: Acyrthisiphon pisum Harris (pea aphid);Aphis craccivora Koch (cowpea aphid); A. fabae Scopoli (black beanaphid); A. gossypii Glover (cotton aphid, melon aphid); A. maidiradicisForbes (corn root aphid); A. pomi De Geer (apple aphid); A. spiraecolaPatch (spirea aphid); Aulacorthum solani Kaltenbach (foxglove aphid);Chaetosiphon fragaefolii Cockerell (strawberry aphid); Diuraphis noxiaKurdjumov/Mordvilko (Russian wheat aphid); Dysaphis plantagineaPaaserini (rosy apple aphid); Eriosoma lanigerum Hausmann (woolly appleaphid); Brevicoryne brassicae Linnaeus (cabbage aphid); Hyalopteruspruni Geoffroy (mealy plum aphid); Lipaphis erysimi Kaltenbach (turnipaphid); Metopolophium dirrhodum Walker (cereal aphid); Macrosiphumeuphorbiae Thomas (potato aphid); Myzus persicae Sulzer (peach potatoaphid, green peach aphid); Nasonovia ribisnigri Mosley (lettuce aphid);Pemphigus spp. (root aphids and gall aphids); Rhopalosiphum maidis Fitch(corn leaf aphid); R. padi Linnaeus (bird cherry-oat aphid); Schizaphisgraminum Rondani (greenbug); Sipha flava Forbes (yellow sugarcaneaphid); Sitobion avenae Fabricius (English grain aphid); Therioaphismaculata Buckton (spotted alfalfa aphid); Toxoptera aurantii Boyer deFonscolombe (black citrus aphid) and T. citricida Kirkaldy (brown citrusaphid); Melanaphis sacchari (sugarcane aphid); Adelges spp. (adelgids);Phylloxera devastatrix Pergande (pecan phylloxera); Bemisia tabaciGennadius (tobacco whitefly, sweetpotato whitefly); B. argentifoliiBellows & Perring (silverleaf whitefly); Dialeurodes citri Ashmead(citrus whitefly); Trialeurodes abutiloneus (bandedwinged whitefly) andT. vaporariorum Westwood (greenhouse whitefly); Empoasca fabae Harris(potato leafhopper); Laodelphax striatellus Fallen (smaller brownplanthopper); Macrolestes quadrilineatus Forbes (aster leafhopper);Nephotettix cinticeps Uhler (green leafhopper); N. nigropictus Stal(rice leafhopper); Nilaparvata lugens Stal (brown planthopper);Peregrinus maidis Ashmead (corn planthopper); Sogatella furciferaHorvath (white backed planthopper); Sogatodes orizicola Muir (ricedelphacid); Typhlocyba pomaria McAtee (white apple leafhopper);Erythroneoura spp. (grape leafhoppers); Magicicada septendecim Linnaeus(periodical cicada); Icerya purchasi Maskell (cottony cushion scale);Quadraspidiotus perniciosus Comstock (San Jose scale); Planococcus citriRisso (citrus mealybug); Pseudococcus spp. (other mealybug complex);Cacopsylla pyricola Foerster (pear psylla); Trioza diospyri Ashmead(persimmon psylla).

Agronomically important species of interest from the order Hemipterainclude, but are not limited to: Acrosternum hilare Say (green stinkbug); Anasa tristis De Geer (squash bug); Blissus leucopterusleucopterus Say (chinch bug); Corythuca gossypii Fabricius (cotton lacebug); Cyrtopeltis modesta Distant (tomato bug); Dysdercus suturellusHerrich-Schaffer (cotton stainer); Euschistus servus Say (brown stinkbug); E. variolarius Palisot de Beauvais (one spotted stink bug);Graptostethus spp. (complex of seed bugs); Leptoglossus corculus Say(leaf footed pine seed bug); Lygus lineolaris Palisot de Beauvais(tarnished plant bug); L. Hesperus Knight (Western tarnished plant bug);L. pratensis Linnaeus (common meadow bug); L. ruguhpennis Poppius(European tarnished plant bug); Lygocoris pabulinus Linnaeus (commongreen capsid); Nezara viridula Linnaeus (southern green stink bug);Oebalus pugnax Fabricius (rice stink bug); Oncopeltus fasciatus Dallas(large milkweed bug); Pseudatomoscelis seriatus Reuter (cotton fleahopper).

Furthermore, embodiments may be effective against Hemiptera such,Calocoris norvegicus Gmelin (strawberry bug); Orthops campestrisLinnaeus; Plesiocoris rugicollis Fallen (apple capsid); Cyrtopeltismodestus Distant (tomato bug); Cyrtopeltis notatus Distant (suckfly);Spanagonicus albofasciatus Reuter (whitemarked fleahopper); Diaphnocorischlorionis Say (honeylocust plant bug); Labopidicola allii Knight (onionplant bug); Pseudatomoscelis seriatus Reuter (cotton fleahopper);Adelphocoris rapidus Say (rapid plant bug); Poecilocapsus lineatusFabricius (four lined plant bug); Nysius ericae Schilling (false chinchbug); Nysius raphanus Howard (false chinch bug); Nezara viridulaLinnaeus (Southern green stink bug); Eurygaster spp.; Coreidae spp.;Pyrrhocoridae spp.; Tinidae spp.; Blostomatidae spp.; Reduviidae spp.and Cimicidae spp.

Also included are adults and larvae of the order Acari (mites) such asAceria tosichella Keifer (wheat curl mite); Petrobia latens Muller(brown wheat mite); spider mites and red mites in the familyTetranychidae, Panonychus ulmi Koch (European red mite); Tetranychusurticae Koch (two spotted spider mite); (T. mcdanieli McGregor (McDanielmite); T. cinnabarinus Boisduval (carmine spider mite); T. turkestaniUgarov & Nikolski (strawberry spider mite); flat mites in the familyTenuipalpidae, Brevipalpus lewisi McGregor (citrus flat mite); rust andbud mites in the family Eriophyidae and other foliar feeding mites andmites important in human and animal health, i.e., dust mites in thefamily Epidermoptidae, follicle mites in the family Demodicidae, grainmites in the family Glycyphagidae, ticks in the order Ixodidae. Ixodesscapularis Say (deer tick); I. holocyclus Neumann (Australian paralysistick); Dermacentor variabilis Say (American dog tick); Amblyommaamericanum Linnaeus (lone star tick) and scab and itch mites in thefamilies Psoroptidae, Pyemotidae and Sarcoptidae.

Insect pests of the order Thysanura are of interest, such as Lepismasaccharina Linnaeus (silverfish); Thermobia domestica Packard(firebrat).

Additional arthropod pests covered include: spiders in the order Araneaesuch as Loxosceles reclusa Gertsch and Mulaik (brown recluse spider) andthe Latrodectus mactans Fabricius (black widow spider) and centipedes inthe order Scutigeromorpha such as Scutigera coleoptrata Linnaeus (housecentipede).

Insect pests of interest include the superfamily of stink bugs and otherrelated insects including but not limited to species belonging to thefamily Pentatomidae (Nezara viridula, Halyomorpha halys, Piezodorusguildini, Euschistus servus, Acrosternum hilare, Euschistus heros,Euschistus tristigmus, Acrosternum hilare, Dichelops furcatus, Dichelopsmelacanthus, and Bagrada hilaris (Bagrada Bug)), the family Plataspidae(Megacopta cribraria-Bean plataspid) and the family Cydnidae(Scaptocoris castanea-Root stink bug) and Lepidoptera species includingbut not limited to: diamondback moth, e.g., Helicoverpa zea Boddie;soybean looper, e.g., Pseudoplusia includens Walker and velvet beancaterpillar e.g., Anticarsia gemmatalis Hubner.

Methods for measuring pesticidal activity are well known in the art.See, for example, Czapla and Lang, (1990) J. Econ. Entomol.83:2480-2485; Andrews, et al., (1988) Biochem. J. 252:199-206; Marrone,et al., (1985) J. of Economic Entomology 78:290-293 and U.S. Pat. No.5,743,477, all of which are herein incorporated by reference in theirentirety. Generally, the protein is mixed and used in feeding assays.See, for example Marrone, et al., (1985) J. of Economic Entomology78:290-293. Such assays can include contacting plants with one or morepests and determining the plant's ability to survive and/or cause thedeath of the pests.

Nematodes include parasitic nematodes such as root-knot, cyst and lesionnematodes, including Heterodera spp., Meloidogyne spp. and Globoderaspp.; particularly members of the cyst nematodes, including, but notlimited to, Heterodera glycines (soybean cyst nematode); Heteroderaschachtii (beet cyst nematode); Heterodera avenae (cereal cyst nematode)and Globodera rostochiensis and Globodera pailida (potato cystnematodes). Lesion nematodes include Pratylenchus spp.

In some aspects, the taught insecticidal proteins are active against aninsect that is resistant to a Cry protein. For example, the taughtinsecticidal proteins may be active against an insect that is resistantto mCry3A, Cry3Bb1, eCry3.1Ab, and the binary protein complexCry34Ab1/Cry3 5Ab1. In aspects, the taught insecticidal proteins areactive against a western corn rootworm (WCR, Diabrotica virgiferavirgifera LeConte) that is resistant to a Cry protein (e.g. Cry3Bb1protein expressed by MON88017). In aspects, the taught insecticidalproteins are active against a western corn rootworm (WCR, Diabroticavirgifera virgifera LeConte) that is resistant to a Cry protein (e.g.mCry3A). In aspects, the taught insecticidal proteins can be toxic tothe corn rootworms of Diabrotica barberi and Diabrotica undecimpunctatahowardi and other beetle species such as Diabrotica speciosa andPhyllotreta cruciferae. In aspects, the taught insecticidal proteins arenot toxic to spotted lady beetle (Coleomegilla maculata) or certainLepidopterans or certain Hemipterans. See, U. Schellenberger et al., “Aselective insecticidal protein from Pseudomonas for controlling cornrootworms,” Science, 2016 Nov. 4; 354(6312):634-637 (providing IPD072Aa,an 86 AA protein, GenBank Accession No. KT795291) incorporated byreference herein; and Jun-Zhi Wei et al., “A selective insecticidalprotein from Pseudomonas mosselii for corn rootworm control,” PlantBiotechnology Journal, 2018, Vol. 16, pgs. 649-659 (providing PIP-47aa)incorporated by reference herein.

Seed Treatment

To protect and to enhance yield production and trait technologies, seedtreatment options can provide additional crop plan flexibility and costeffective control against insects, weeds and diseases. Seed material canbe treated, typically surface treated, with a composition comprisingcombinations of chemical or biological herbicides, herbicide safeners,insecticides, fungicides, germination inhibitors and enhancers,nutrients, plant growth regulators and activators, bactericides,nematocides, and/or molluscicides.

These compounds are typically formulated together with further carriers,surfactants or application promoting adjuvants customarily employed inthe art of formulation. The coatings may be applied by impregnatingpropagation material with a liquid formulation or by coating with acombined wet or dry formulation. Examples of the various types ofcompounds that may be used as seed treatments are provided in ThePesticide Manual: A World Compendium, C. D. S. Tomlin Ed., Published bythe British Crop Production Council, which is hereby incorporated byreference.

Some seed treatments that may be used on crop seed include, but are notlimited to, one or more of abscisic acid, acibenzolar-S-methyl,avermectin, amitrol, azaconazole, azospirillum, azadirachtin,azoxystrobin, Bacillus spp. (including one or more of cereus, firmus,megaterium, pumilis, sphaericus, subtilis and/or thuringiensis species),bradyrhizobium spp. (including one or more of betae, canariense,elkanii, iriomotense, japonicum, liaonigense, pachyrhizi and/oryuanmingense), captan, carboxin, chitosan, clothianidin, copper,cyazypyr, difenoconazole, etidiazole, fipronil, fludioxonil,fluoxastrobin, fluquinconazole, flurazole, fluxofenim, harpin protein,imazalil, imidacloprid, ipconazole, isoflavenoids,lipo-chitooligosaccharide, mancozeb, manganese, maneb, mefenoxam,metalaxyl, metconazole, myclobutanil, PCNB, penflufen, penicillium,penthiopyrad, permethrine, picoxystrobin, prothioconazole,pyraclostrobin, rynaxypyr, S-metolachlor, saponin, sedaxane, TCMTB,tebuconazole, thiabendazole, thiamethoxam, thiocarb, thiram,tolclofos-methyl, triadimenol, trichoderma, trifloxystrobin,triticonazole and/or zinc. PCNB seed coat refers to EPA RegistrationNumber 00293500419, containing quintozen and terrazole. TCMTB refers to2-(thiocyanomethylthio) benzothiazole.

Seed varieties and seeds with specific transgenic traits may be testedto determine which seed treatment options and application rates maycomplement such varieties and transgenic traits in order to enhanceyield. For example, a variety with good yield potential but head smutsusceptibility may benefit from the use of a seed treatment thatprovides protection against head smut, a variety with good yieldpotential but cyst nematode susceptibility may benefit from the use of aseed treatment that provides protection against cyst nematode, and soon. Likewise, a variety encompassing a transgenic trait conferringinsect resistance may benefit from the second mode of action conferredby the seed treatment, a variety encompassing a transgenic traitconferring herbicide resistance may benefit from a seed treatment with asafener that enhances the plants resistance to that herbicide, etc.Further, the good root establishment and early emergence that resultsfrom the proper use of a seed treatment may result in more efficientnitrogen use, a better ability to withstand drought and an overallincrease in yield potential of a variety or varieties containing acertain trait when combined with a seed treatment.

Methods for Killing an Insect Pest and Controlling an Insect Population

In some embodiments, methods are provided for killing an insect pest,comprising contacting the insect pest with an insecticidally effectiveamount of a recombinant protein as taught herein. In some embodiments,methods are provided for killing an insect pest, comprising contactingthe insect pest with an insecticidally effective amount of a pesticidalprotein of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18,SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO:28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ IDNO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56,SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO:66, SEQ ID NO: 68, SEQ ID NO: 70, or SEQ ID NO: 72, or a variantthereof.

In some embodiments, methods are provided for controlling an insect pestpopulation, comprising contacting the insect pest population with aninsecticidally effective amount of a recombinant protein as taughtherein. In some embodiments, methods are provided for controlling aninsect pest population, comprising contacting the insect pest populationwith an insecticidally effective amount of a pesticidal protein of SEQID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20,SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO:30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ IDNO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58,SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO:68, SEQ ID NO: 70, or SEQ ID NO: 72, or a variant thereof.

As used herein, “controlling a pest population” or “controls a pest”refers to any effect on a pest that results in limiting the damage thatthe pest causes. Controlling a pest includes, but is not limited to,killing the pest, inhibiting development of the pest, altering fertilityor growth of the pest in such a manner that the pest provides lessdamage to the plant, decreasing the number of offspring produced,producing less fit pests, producing pests more susceptible to predatorattack or deterring the pests from eating the plant.

In some embodiments, methods are provided for controlling an insect pestpopulation resistant to a pesticidal protein, comprising contacting theinsect pest population with an insecticidally effective amount of arecombinant protein as taught herein. In some embodiments, methods areprovided for controlling an insect pest population resistant to apesticidal protein, comprising contacting the insect pest populationwith an insecticidally effective amount of a recombinant pesticidalprotein of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18,SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO:28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ IDNO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56,SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO:66, SEQ ID NO: 68, SEQ ID NO: 70, or SEQ ID NO: 72, or a variantthereof.

In some embodiments, methods are provided for protecting a plant from aninsect pest, comprising expressing in the plant or cell thereof arecombinant polynucleotide encoding a pesticidal protein as taughtherein. In some embodiments, methods are provided for protecting a plantfrom an insect pest, comprising expressing in the plant or cell thereofa recombinant polynucleotide encoding a pesticidal protein of SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ IDNO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO:50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ IDNO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQID NO: 70, or SEQ ID NO: 72, or a variant thereof.

Methods for Increasing Plant Yield

Methods for increasing plant yield are provided. The methods compriseproviding a plant or plant cell expressing a polynucleotide encoding thepesticidal polypeptide sequence disclosed herein and growing the plantor a seed thereof in a field infested with a pest against which thepolypeptide has pesticidal activity. In some embodiments, thepolypeptide has pesticidal activity against a Lepidopteran, Coleopteran,Dipteran, Hemipteran or nematode pest, and the field is infested with aLepidopteran, Hemipteran, Coleopteran, Dipteran or nematode pest.

As defined herein, the “yield” of the plant refers to the quality and/orquantity of biomass produced by the plant. “Biomass” as used hereinrefers to any measured plant product. An increase in biomass productionis any improvement in the yield of the measured plant product.Increasing plant yield has several commercial applications. For example,increasing plant leaf biomass may increase the yield of leafy vegetablesfor human or animal consumption. Additionally, increasing leaf biomasscan be used to increase production of plant derived pharmaceutical orindustrial products. An increase in yield can comprise any statisticallysignificant increase including, but not limited to, at least a 1%increase, at least a 3% increase, at least a 5% increase, at least a 10%increase, at least a 20% increase, at least a 30%, at least a 50%, atleast a 70%, at least a 100% or a greater increase in yield compared toa plant not expressing the pesticidal sequence. In specific methods,plant yield is increased as a result of improved pest resistance of aplant expressing an insecticidal protein disclosed herein.

Hidden Markov Model

A hidden Markov model (HMM) is a statistical model that can be used todescribe the evolution of observable events that depend on internalfactors, which are not directly observable. The observed event is calleda “symbol” and the invisible factor underlying the observation a“state”. An HMM consists of two stochastic processes, namely, aninvisible process of hidden states and a visible process of observablesymbols. The hidden states form a Markov chain, and the probabilitydistribution of the observed symbol depends on the underlying state. Forthis reason, an HMI is also called a doubly-embedded stochastic process.Modeling observations in these two layers, one visible and the otherinvisible, is very useful, since many real world problems deal withclassifying raw observations into a number of categories, or classlabels, which are more meaningful. This approach is useful in modelingbiological sequences, such as proteins and DNA sequences. Typically, abiological sequence consists of smaller substructures with differentfunctions, and different functional regions often display distinctstatistical properties. For example, it is well known that proteinsgenerally consist of multiple domains. Given a new protein, HMMs can beused to predict the constituting domains (corresponding to one or morestates in an HMM) and their locations in the amino acid sequence(observations). Furthermore, we may also want to find the protein familyto which this new protein sequence belongs. In fact, HMIs have beenshown to be very effective in representing biological sequences. As aresult, HMIs have become increasingly popular in computational molecularbiology, bioinformatics, and many state-of-the-art sequence analysisalgorithms have been built on HMIs. See, Byung-Jun Yoon, “Hidden MarkovModels and Their Applications in Biological Sequence Analysis,” CurrentGenomics, 2009, Vol. 10, pgs. 402-415, for a comprehensive review, saidarticle is incorporated herein by reference.

Thus, it is understood that a Markov model is a system that produces aMarkov chain, and a hidden Markov model is one where the rules forproducing the chain are unknown or “hidden.” The rules include twoprobabilities: (i) that there will be a certain observation and (ii)that there will be a certain state transition, given the state of themodel at a certain time. The Hidden Markov Model (HMM) method is amathematical approach to solving certain types of problems: (i) giventhe model, find the probability of the observations; (ii) given themodel and the observations, find the most likely state transitiontrajectory; and (iii) maximize either i or ii by adjusting the model'sparameters. For each of these problems, algorithms have been developed,for example: (i) Forward-Backward, (ii) Viterbi, and (iii) Baum-Welch(and the Segmental K-means alternative), among others

HMMER Software

HMMER is a HMM software package that is used to search sequencedatabases for homologs of protein or DNA sequences, and to make sequencealignments. HMMER can be used to search sequence databases with singlequery sequences, but it becomes particularly powerful when the query isan alignment of multiple instances of a sequence family. HMMER makes aprofile of the query that assigns a position-specific scoring system forsubstitutions, insertions, and deletions. HMMER profiles areprobabilistic models called “profile hidden Markov models” (profileHMMs) (Krogh et al., 1994; Eddy, 1998; Durbin et al., 1998). Compared toBLAST, FASTA, and other sequence alignment and database search toolsbased on older scoring methodology, HMMER aims to be significantly moreaccurate and more able to detect remote homologs, because of thestrength of its underlying probability models.

Profile HMMs are statistical models of multiple sequence alignments, oreven of single sequences. They capture position-specific informationabout how conserved each column of the alignment is, and which residuesare likely. Anders Krogh, David Haussler, and co-workers at UC SantaCruz introduced profile HMMs to computational biology (Krogh et al.,1994), adopting HMM techniques which have been used for years in speechrecognition. HMMs had been used in biology before the Krogh/Hausslerwork, notably by Gary Churchill (Churchill, 1989), but the Krogh paperhad a dramatic impact because HMM technology was so well-suited to thepopular “profile” methods for searching databases using multiplesequence alignments instead of single query sequences. “Profiles” hadbeen introduced by Gribskov and colleagues (Gribskov et al., 1987,1990), and several other groups introduced similar approaches at aboutthe same time, such as “flexible patterns” (Barton, 1990), and“templates” (Bashford et al., 1987; Taylor, 1986). The term “profile”has stuck. All profile methods (including PSI-BLAST (Altschul et al.,1997)) are more or less statistical descriptions of the consensus of amultiple sequence alignment. They use position-specific scores for aminoacids or nucleotides (residues) and position specific penalties foropening and extending an insertion or deletion. Traditional pairwisealignment (for example, BLAST (Altschul et al., 1990), FASTA (Pearsonand Lipman, 1988), or the Smith/Waterman algorithm (Smith and Waterman,1981)) uses position-independent scoring parameters. This property ofprofiles captures important information about the degree of conservationat various positions in the multiple alignment, and the varying degreeto which gaps and insertions are permitted.

The advantage of using HMIs is that HMMs have a formal probabilisticbasis. They use probability theory to guide how all the scoringparameters should be set. For example, HMMs have a consistent theory forsetting position-specific gap and insertion scores. The methods areconsistent and therefore highly automatable, allowing one to makelibraries of hundreds of profile HMMs and apply them on a very largescale to whole genome analysis. One such database of protein domainmodels is Pfam (Sonnhammer et al., 1997; Finn et al., 2010), which is asignificant part of the Interpro protein domain annotation system(Mulder et al., 2003). The construction and use of Pfam is tightly tiedto the HMMER software package.

Insecticidal Protein Discovery Platform (IPDP)

The disclosure presents a platform for discovering novel insecticidalproteins from highly heterogeneous environmental sources. Themethodology utilizes metagenomic enrichment procedures and geneticamplification techniques, which enables access to a broad class ofunknown microbial diversity and their resultant proteome.

FIG. 1 provides an overall workflow illustrating the IPDP, which will bediscussed in detail below.

Collect Soil Samples

1-10 grams of material is collected from an environmental sample thatcontains rich microbial diversity.

Resuspend in Buffered Solution

Environmental material is resuspended and stirred continuously in 10-100mLs of PBS (phosphate buffered solution) for 15 minutes. Largeparticulates are then allowed to settle.

Dilution and Planting on Solid Nutrient Limiting Agar Media

A series of dilutions of the supernatant is plated on nutrient limitingagar containing cyclohexamide to reduce fungal growth.

Various media recipes have been described in the literature to favorgrowth of particular families of microbes.

The current IPDP utilizes a proprietary media, and media growthprocedure, in order to enrich for microbes of a particular Genus (e.g.Pseudomonas in certain embodiments).

Collect Bacterial Growth from Plates—Isolate all Genomic DNA fromEnriched Sample as a Mixture Via Lysis and Precipitation

All bacterial growth on plates is collected by washing with water, cellsare pelleted by centrifugation, and the supernatant is discarded.

Genomic DNA was isolated from the pelleted metagenomics sample usingstandard bacterial genomic DNA isolation techniques.

Genes Encoding Proteins from the Monalysin Class are Further EnrichedUsing Degenerate PCR and Cloned into Plasmid Vectors for Recovery andSequencing

Proprietary degenerate primers were utilized to amplify genes encodingproteins from the monalysin class from the metagenomic DNA sample.

A “monalysin class” of protein can be a protein that has a degree ofsimilarity to, e.g. SEQ ID NO: 87, from Table 1. The present IPDP has asubstantial library of proprietary degenerate primers, which can beutilized to search for proteins in this class.

Amplified DNA of ˜800 bp in size were separated by gel electrophoresisand recovered utilizing standard techniques.

The degenerate primers include tails compatible for cloning into a DNAplasmid. The PCR-amplified DNA were cloned and sequenced to identifyfull-length genes encoding proteins with similarity to the publishedmonalysin sequence from Opota et al.

The combination of: 1) an initial enrichment of certain microbialpopulations on nutrient limited agar, followed by 2) amplification ofgenes encoding monalysin-class proteins, using degenerate PCR from thegenomic DNA isolated from the enriched population, are both preferredsteps in some embodiments, to recover genes encoding monalysin-likeproteins.

“Monalysin-like” proteins can be defined as proteins that have somedegree of similarity to the monalysin protein described in Opota, etal., See Opota, et al., “Monalysin, a Novel B-Pore-Forming Toxin fromthe Drosophila Pathogen Pseudomonas entomophila, Contributes to HostIntestinal Damage and Lethality,” PLoS Pathogens, September 2011, Vol.7, Issue 9 (incorporated herein by reference). The terms“monalysin-like” and “monalysin class” of protein are usedinterchangeably. Furthermore, as aforementioned, the current applicationprovides the sequence for the monalysin described in Opota in Table 1,and SEQ ID NO: 87.

Genomic DNA collected from bacteria isolated on rich media from theoriginal environmental sample did not yield any amplified product usingdegenerate PCR. Thus, the nutrient limited agar (developed to enrich formicrobes of a particular Genus) step was successful in allowing the IPDPto access microbial organisms that are often not available to currentmethods in the art. Furthermore, sequencing of the enriched genomic DNAdid not yield the number of sequences that were eventually obtainedutilizing the above described combined approach (i.e. enrichment anddegenerate PCR amplification), suggesting the discovered insecticidalprotein sequences are quite rare, even in the enriched populations, andthe amplification step following enrichment is preferred in someaspects.

Identification of Novel Insecticidal Proteins Utilizing Homology andProfile/HMM Methods

The IPDP can optionally involve the use of an HMM to identifyinsecticidal proteins. An HMM profile built based on known insecticidalproteins (e.g., an HMM built based on known monalysins) can be used toscan the enriched DNA library for genes which encode proteins with aminoacid sequences which score highly when analyzed using the HMI profile.Additionally or alternatively, new insecticidal proteins can beidentified by comparing sequences in the enriched DNA library tosequences encoding known insecticidal proteins in genomic databases,e.g., using sequence analysis tools like BLAST and searching for mutualbest hit sequences against sequences in GENBANK.

An example of the HMI process is described in Example 5 and an exampleHMM built using insecticidal proteins identified using methods describedherein is provided in Table 6. The HMM was built using eightinsecticidal proteins discovered via the IPDP and found in Table 3.These proteins have the amino acid sequences shown in: a) SEQ ID NO: 2that is ZIP1, b) SEQ ID NO: 4 that is ZIP2, c) SEQ ID NO: 12 that isZIP6, d) SEQ ID NO: 14 that is ZIP5, e) SEQ ID NO: 16 that is ZIP5, f)SEQ ID NO: 18 that is ZIP10, g) SEQ ID NO: 20 that is ZIP11, and h) SEQID NO: 22 that is ZIP12. To discover new insecticidal proteins, anenriched genomic DNA library built using the methods disclosed hereincan be searched using the HMM provided in Table 6. Sequences whichreceive a high score based on that comparison can be identified as newinsecticidal proteins. In certain embodiments, sequences receiving ahigh score are those sequences which score at or above a bit score of521.5 and/or sequences which match with an E-value of less than or equalto 7.9e-161 when scored using the HMM in Table 6.

Thus, in certain embodiments, the disclosure provides novel insecticidalproteins, the proteins having an amino acid sequence which score at orabove a bit score of 521.5 and/or sequences which match at an E-value ofless than or equal to 7.9e-161 when scored using the HMM in Table 6.These proteins can be provided in any form (e.g., as isolated orrecombinant proteins) or as part of any of the compositions (e.g.,plants or agricultural compositions) disclosed herein.

Examples

The following examples are given for the purpose of illustrating variousembodiments of the disclosure and are not meant to limit the presentdisclosure in any fashion. Changes therein and other uses which areencompassed within the spirit of the disclosure, as defined by the scopeof the claims, will be recognized by those skilled in the art.

A brief table of contents is provided below solely for the purpose ofassisting the reader. Nothing in this table of contents is meant tolimit the scope of the examples or disclosure of the application.

TABLE 2 Table of Contents For Example Section Example Title BriefDescription 1 Insecticidal Protein Discovery Platform Describesimplementation of the novel (IPDP) insecticidal protein discoveryplatform. 2 Novel Insecticidal Proteins Discovered Describes a selectset of novel with the IPDP insecticidal proteins identified via theIPDP. 3 Insecticidal Proteins-Lysate Insect Describes a lysate feedingassay that Feeding Assays contains an insecticidal protein discoveredvia the IPDP, which shows 100% mortality against an insect pest from thePentatomidae family (i.e. Halyomorpha halys Stål, 1855). 4 InsecticidalProteins-Purified Protein Describes experiments conducted with a InsectFeeding Assays range of assays demonstrating that a purifiedinsecticidal protein, as taught herein, has activity against a host ofinsect species. 5 IPDP-HMM Construction Describes implementation of theIPDP's Hidden Markov Model feature to predict undiscovered insecticidalproteins. 6 Transformed Plants Describes experiments conducteddemonstrating plants transformed to express the insecticidal proteins.

Example 1: Insecticidal Protein Discovery Platform (IPDP)

This example describes an implementation of the IPDP. Further detailsregarding the IPDP can be found in the aforementioned IPDP section,which immediately precedes the Example section.

First, 5 grams of material from an environmental sample rich inmicrobial diversity was collected and stored.

This material was resuspended in 50 mL of PBS and stirred continuouslyfor 15 minutes. After 15 minutes, large particulates were allowed tosettle and serial dilutions of the supernatant were plated on aproprietary nutrient limiting agar media containing cyclohexamide toreduce fungal growth. Plates were grown at 18° C. for 10-14 days.

Bacteria growth on these plates was collected by repeated washing with 4mLs of water and collected in a 15 mL conical tube. Microbial cells werepelleted by centrifugation and the supernatant was discarded.

Genomic DNA was isolated from cell pellets using the Wizard Genomic DNAPurification Kit from Promega.

Proprietary degenerate primers were used to amplify DNA via PCR.Amplicons of −800 bp were gel purified and cloned into a DNA plasmidvector.

Cloned amplicons were sequenced using Sanger sequencing and encodedproteins were compared to the published monalysin sequence (i.e. asdescribed in Opota et al. and found in Table 1, and SEQ ID NO: 87).

Example 2: Novel Insecticidal Proteins Discovered with the IPDP

The aforementioned IPDP from Example 1 was able to identify at least 36novel insecticidal proteins, which are represented in the below Table 3.

From these 36 novel insecticidal proteins, homology based analysis hasrevealed that at least 32 of these proteins have at least a 20% sequenceidentity difference from the known sequence identity of any insecticidalprotein in this class. This is a significant finding, as it demonstratesthe power of the taught IPDP, in finding insecticidal proteins that arenovel over those found in the art.

Of the 32 sequences having at least a 20% sequence identity difference,eight insecticidal proteins that have at least a 20% sequence identitydifference from any known protein in this class were selected forfurther analysis and include: (1) ZIP1, (2) ZIP2, (3) ZIP6, (4) ZIP8,(5) ZIP9, (6) ZIP10, (7) ZIP11, and (8) ZIP12. These proteins sharesignificant homology amongst one another and therefore point toconserved insecticidal domains that could be shared among this novelgroup of insecticidal proteins. The multiple sequence alignment forthese eight proteins can be found in FIG. 3 with a correspondingphylogenetic tree found in FIG. 5.

TABLE 3 Novel Insecticidal Proteins Identified via the IPDP IdentifierNucleotide Amino Acid ZIP1 SEQ ID NO: 1 SEQ ID NO: 2ATGACGATCAAGGAAGAGCTGAGCCAACCCCAAAGCCATTCGGTCG MTIKEELSQPQSHSVELDQAGCTCGACCAGTTGCAAGTCGGGGAAGTCTCTGCACGCGAAGCGTT LQVGEVSAREALTSNFAGSGACCTCCAACTTCGCCGGCAGTTTCGATCAGTTCCCGACCAAAAGC FDQFPTKSGSFEIDKYLLNGGCAGCTTCGAGATCGACAAATACCTGCTCAACTACGCGGATCCGA YADPKKGCWLDGVTVYGDIAAAAAGGCTGCTGGCTGGACGGCGTCACCGTCTACGGTGACATCTA YIGKQNWGTYTRPVFAYLQCATCGGCAAGCAGAACTGGGGCACCTACACGCGCCCGGTGTTCGCC HTDTISIPQQVTQTKSYQLTACCTGCAGCACACCGACACCATCTCGATTCCGCAGCAGGTGACGC SKGHTQSFTKSVSAKYSVGAGACCAAGAGCTACCAGTTGAGCAAAGGCCACACCCAGTCGTTCAC GSIDIVNISSDITVGFSSTCAAGTCGGTCAGCGCCAAATACAGCGTTGGCGGCAGTATCGACATC EAWSTNQTFTQSTELAGPGGTCAACATCAGCTCGGATATCACCGTTGGTTTCAGCAGCACCGAGG TFFVYQVVFVYAHNATSAGCCTGGTCGACGAACCAGACCTTCACCCAAAGCACCGAGCTGGCCGG GQNGNAFAYSKTQQVNSRLCCCTGGCACCTTCTTTGTCTATCAGGTGGTGTTTGTCTATGCGCAC DLYYLSAITQDRTVIVESSAACGCCACTTCGGCCGGTGGGCAGAATGGCAATGCCTTTGCCTATA NAIDPLDWDTVQRNVLIQNGCAAGACCCAGCAGGTGAACTCGCGGCTCGACCTTTACTACCTGTC YNPASNSGHFSFDWSAYNDGGCCATCACCCAGGACCGTACGGTCATCGTCGAGTCCAGCAATGCA PHRRYATCGACCCGCTGGACTGGGATACCGTGCAGCGCAACGTGCTGATCCAGAACTACAACCCGGCCAGCAACAGCGGGCACTTCTCGTTCGACTGGAGCGCCTACAACGATCCTCATCGCCGTTAT ZIP2 SEQ ID NO: 3 SEQ ID NO: 4ATGACGATCAAGGAAGAGCTGAGCCAACCCCAAAGCCATTCGGTCG MTIKEELSQPQSHSVELDQAGCTCGACCAGTTGCAAGTCGGGGAAGTCTCTGCACGCGAAGCGTT LQVGEVSAREALTSNFAGSGACCTCCAACTTCGCCGGCAGTTTCGATCAGTTCCCGACCAAAAGC FDQFPTKSGSFEIDKYLLNGGCAGCTTCGAGATCGACAAATACCTGCTCAACTACGCGGATCCGA YADPKKGCWLDGVTVYGDIAAAAAGGCTGCTGGCTGGACGGCGTCACCGTCTACGGTGACATCTA YIGKQNWGTYTRPVFAYLQCATCGGCAAGCAGAACTGGGGCACCTACACGCGCCCGGTGTTCGCC HTDTISIPQQVTQTKSYQLTACCTGCAGCACACCGACACCATCTCGATTCCGCAGCAGGTGACGC SKGHTQSFTKSVSAKYSVGAGACCAAGAGCTACCAGTTGAGCAAAGGCCACACCCAGTCGTTCAC GSIDIVNISSDITVGFSSTCAAGTCGGTCAGCGCCAAATACAGCGTTGGCGGCAGTATCGACATC EAWSTNQTFTQSTELAGPGGTCAACATCAGCTCGGATATCACCGTTGGTTTCAGCAGCACCGAGG TFFVYQVVFVYAHNATSAGCCTGGTCGACGAACCAGACCTTCACCCAAAGCACCGAGCTGGCCGG GQNGNAFAYSKTQQVNSRLCCCTGGCACCTTCTTTGTCTATCAGGTGGTGTTTGTCTATGCGCAC DLYYLSAITQDRTVIVESSAACGCCACTTCGGCCGGTGGGCAGAATGGCAATGCCTTTGCCTATA NAIDPLDWDTVQRNVLIQNGCAAGACCCAGCAGGTGAACTCGCGGCTCGACCTTTACTACCTGTC YNPASNSGHFSFDWSAYTIGGCCATCACCCAGGACCGTACGGTCATCGTCGAGTCCAGCAATGCA LIAVIELRSGCATCGACCCGCTGGACTGGGATACCGTGCAGCGCAACGTGCTGATCCAGAACTACAACCCGGCCAGCAACAGCGGGCACTTCTCGTTCGACTGGAGCGCCTACACGATCCTCATCGCCGTTATTGAACTGAGATCCGGC TGC ZIP3 SEQ ID NO: 5SEQ ID NO: 6 ATGACGATCAAGGAAGAGCTGAGCCAACCCCAAAGCCATTCGGTCGMTIKEELSQPQSHSVELDQ AGCTCGACCAGTTGCAAGTCGGGGAAGTCCCTGCACGCGAAGCGTTLQVGEVPAREALTSNFAGS GACCTCCAACTTCGCCGGCAGTTTCGATCAGTTCCCGACCAAAAGCFDQFPTKSGSFEIDKYLLN GGCAGCTTCGAGATCGACAAATACCTGCTCAACTACGCGGATCCGAYADPKKGCWLDGVTVYGDI AAAAAGGCTGCTGGCTGGACGGCGTCACCGTCTACGGTGACATCTAYIGKQNWGTYTRPVFAYLQ CATCGGCAAGCAGAACTGGGGCACCTACACGCGCCCGGTGTTCGCCHTDTISIPQQETQTKSYQL TACCTGCAGCACACCGACACCATCTCGATTCCGCAGCAGGAGACGCSKGHTQSFTKSVSAKYGVG AGACCAAGAGCTACCAGTTGAGCAAAGGCCACACCCAGTCGTTCACGSIDIVNISSDITVGFSST CAAGTCGGTCAGCGCCAAATACGGCGTTGGCGGCAGTATCGACATCEAWSTNQTFTQSTELAGPG GTCAACATCAGCTCGGATATCACCGTTGGTTTCAGCAGCACCGAGGTFFVYQVVLVYAHNATSAG CCTGGTCGACGAACCAGACCTTCACCCAAAGCACCGAGCTGGCCGGRQNGNAFAYNKTQQVGSRL CCCTGGCACCTTCTTTGTCTATCAGGTTGTTCTTGTTTATGCGCACDLYYLSAITQNRTVIVESS AACGCCACTTCGGCGGGCAGGCAGAATGGTAATGCCTTCGCCTATAKAIDPLDWDTVQRNVLMEN ACAAGACCCAGCAGGTGGGCTCGCGCCTGGACCTGTACTACCTGTCYNPASNSGHFSFDWSAYND GGCCATCACTCAGAACAGAACGGTCATTGTCGAGTCCAGCAAGGCC PHRRYATCGACCCGCTGGATTGGGATACGGTGCAACGCAACGTGCTGATGGAAAACTACAACCCAGCCAGTAACAGCGGACACTTCAGCTTCGACTGGAGTGCCTACAACGATCCTCATCGCCGTTAT ZIP4 SEQ ID NO: 7 SEQ ID NO: 8ATGACGATCAAGGAAGAACTGAGCAATCCTCAAAGCCATTCGGTCG MTIKEELSQPQSHSVELDQAGCTCGACCAGTTGCAAGTCGGGGAAGTCTCTGCACGCGAAGCGTT LQVGEVSAREALTSNFAGSGACCGCCAACTTCGCCGGCAGTTTCGATCAGTTCCCGACCAAAAGC FDQFPTKSGSFEIDKYLLNGGCAGCTTCGAGATCGACAAATACCTGCTCAACTACGCAGACCCGA YADPKKGCWLDGVTVYGDIAACAAGGCTGCTGGCTGGACGGCGTCACCGTCTACGGTGACATCTA YIGKQNWGTYTRPVFAYLQCATCGGCAAGCAGAACTGGGGCACCTACACGCGCCCGGTGTTCGCC YVETISIPQNVTTTLSYQLTACCTGCAGCACACGGACACCATCTCGATTCCGCAGCAGGTGACGC TKGHTRSFETSVNAKYSVGAGACCAAGAGCTACCAGTTGAGCAAAGGCCACACCCAGTCGTTCAC ANIDIVNVGSEISTGFTRSCAAGTCGGTCAGCGCCAAGTACAGCGTTGGCGGCAGTATCGACATC ESWSTTQSFTDTTEMKGPGGTCAACGTCAGCTCGGATATCACCGTTGGTTTCAGCAGCACCGAGG TFVIYQVVLVYAHNATSAGCCTGGTCGACGACCCAGACCTTCACCCAGAGCACCGAGCTGGCCGG RQNANAFAYSKTQAVGSRVCCCTGGCACCTTCTTTGTCTATCAGGTGGTGTTTGTCTACGCGCAC DLYYLSAITQRKRVIVPSSAACGCCACCTCGGCGGGCCGGCAGAATGGCAATGCCTTTGCCTATA NAVTPLDWDTVQRNVLMENGCAAGACCCAGCAGGTGGATTCGCGGCTCGATCTCTACTATCTGTC YNPXSNSGHFSFDWSAYNDGCGATCACCCAGGACCGTACGGTCATCGTCGAGTCCAGCAAGGCAA PHRRYTCAACCCGCTGGACTGGGATACCGTGCAGCGCAACGTGCTGATCGAGAACTACAACCCGGCCTCCAACAGTGGGCACTTCCGCTTCGACTGGAGCGCCTACAACGATCCTCATCGTCGTTAC ZIP5 SEQ ID NO: 9 SEQ ID NO: 10ATGACGATCAAGGAAGAGCTGAGCAATCCTCAAAGCCATTCGGTCG MTIKEELSNPQSHSVELDQAGCTCGACCAGTTGCAAGTCGGGGAAGTCTCTGCACGCGAAGCGTT LQVGEVSAREALTANFAGSGACCGCCAACTTCGCCGGCAGTTTCGATCAGTTCCCGACCAAAAGC FDQFPTKSGSFEIDKYLLNGGCAGCTTCGAGATCGACAAATACCTGCTCAACTACGCAGACCCGA YADPKQGCWLDGVTVYGDIAACAAGGCTGCTGGCTGGACGGCGTCACCGTCTACGGTGACATCTA YIGKQNWGTYTRPVFAYLQCATCGGCAAGCAGAACTGGGGCACCTACACGCGCCCGGTGTTCGCC HTDTISIPQQVTQTKSYQLTACCTGCAACACACGGACACCATCTCGATTCCGCAGCAGGTGACGC SKGHTQSFTKSVSAKYNVGAGACCAAGAGCTACCAGTTGAGCAAAGGCCACACCCAGTCGTTCAC GSIDIVNVSSDITVGFSSTCAAGTCGGTCAGCGCCAAGTACAACGTTGGCGGCAGTATCGACATC EAWSTTQTFTQSTELAGPGGTCAACGTCAGCTCGGATATCACTGTCGGTTTCAGCAGCACCGAGG TFFVYQVVFVYAHNATSAGCCTGGTCGACGACCCAGACCTTCACCCAGAGCACCGAGCTGGCCGG RQNGNAFAYSKTQQVDSRLCCCTGGCACCTTCTTTGTCTATCAGGTGGTGTTTGTCTACGCGCAC DLYYLSAITQDRTVIVESSAACGCCACCTCGGCGGGCCGGCAGAATGGCAATGCCTTTGCCTATA KAINPLDRDTVQRNVLIENGCAAGACCCAGCAGGTGGATTCGCGGCTCGATCTCTACTACCTGTC YNPASNSGHFRFDWSAYNDGGCCATCACCCAGGACCGTACGGTCATCGTCGAGTCCAGCAAGGCA PHRRYATCAACCCGCTGGACCGGGATACCGTGCAGCGCAACGTGCTGATCGAGAACTACAACCCGGCCTCCAACAGTGGGCACTTCCGCTTCGACTGGAGCGCCTACAACGATCCTCATCGCCGTTAT ZIP6 SEQ ID NO: 11 SEQ ID NO: 12ATGACGATCAAGGAAGAGCTGGGCCAACCCCAAAGCCATTCGGTCG MTIKEELGQPQSHSVELDQAGCTCGACCAGTTGCAAGTCGGGGAAGTCTCTGCACGCGAAGCGTT LQVGEVSAREALTSNFAGSGACCTCCAACTTCGCCGGCAGTTTCGATCAGTTCCCGACCAAAAGC FDQFPTKSGSFEIDKYLLNGGCAGCTTCGAGATCGACAAATACCTGCTCAACTACGCGGATCCGA YADPKKGCWLDGVTVYGDIAAAAAGGCTGCTGGCTGGACGGCGTCACCGTCTACGGTGACATCTA YIGKQNWGTYTRPVSAYLQCATCGGCAAGCAGAACTGGGGCACCTACACGCGCCCGGTGTCCGCC HTDTISIPQQVTQTKSYQLTACCTGCAGCACACCGACACCATCTCGATTCCGCAGCAGGTGACGC SKGHTQSFTKSVSAKYSVGAGACCAAGAGCTACCAGTTGAGCAAAGGCCACACCCAGTCGTTCAC GSIDIVNISSDITVGFSSTCAAGTCGGTCAGCGCCAAATACAGCGTTGGCGGCAGTATCGACATC EAWSTNQTFTQSTELAGPGGTCAACATCAGCTCGGATATCACCGTTGGTTTCAGCAGCACCGAGG TFFVYQVVFVYAHNATSAGCCTGGTCGACGAACCAGACCTTCACCCAAAGCACCGAGCTGGCCGG GQNGNAFAYSKTQQVNSRLCCCTGGCACCTTCTTTGTCTATCAGGTGGTGTTTGTCTATGCGCAC DLYYLSAITQDRTVIVESSAACGCCACTTCGGCCGGTGGGCAGAATGGCAATGCCTTTGCCTATA KAIAPLDWDTVQRNVLMENGCAAGACCCAGCAGGTGAACTCGCGGCTCGACCTTTACTACCTGTC YNQSSNSGHFSFDWSAYNDGGCCATCACCCAGGACCGTACGGTCATCGTCGAGTCCAGCAAGGCC PHRRYATCGCGCCGCTGGATTGGGATACTGTCCAGCGCAATGTACTGATGGAGAACTACAACCAGAGCAGCAATAGCGGGCACTTCAGTTTCGACTGGAGCGCCTACAACGATCCTCATCGCCGTTAT ZIP8 SEQ ID NO: 13 SEQ ID NO: 14ATGACGATCAAGGAAGAGCTGAGCCAACCCCAAAGCCATTCGGTCG MTIKEELSQPQSHSVELDQAGCTCGACCAGTTGCAAGTCGGGGAAGTCTCTGCACGCGAAGCGTT LQVGEVSAREALTSNFAGSGACCTCCAACTTCGCCGGCAGTTTCGATCAGTTCCCGACCAAAAGC FDQFPTKSGSFEIDKYLLNGGCAGCTTCGAGATCGACAAATACCTGCTCAACTACGCGGATCCGA YADPKKGCWLDGVTVYGDIAAAAAGGCTGCTGGCTGGACGGCGTCACCGTCTACGGTGACATCTA YIGKQNWGTYTRPVFAYLQCATCGGCAAGCAGAACTGGGGCACCTACACGCGCCCGGTGTTCGCC HTDTILIPQQVTQTKSYQLTACCTGCAGCACACCGACACCATCTTGATTCCGCAGCAGGTGACGC SKGHTQSFTKSVSAKYSVGAGACCAAGAGCTACCAGTTGAGCAAAGGCCACACCCAGTCGTTCAC GSIDIVNISSDITVGFSSTCAAGTCGGTCAGCGCCAAATACAGCGTTGGCGGCAGTATCGACATC EAWSTNQTFTQSTELAGPGGTCAACATCAGCTCGGATATCACCGTTGGTTTCAGCAGCACCGAGG TFFVYQVVFVYAHNATSAGCCTGGTCGACGAACCAGACCTTCACCCAAAGCACCGAGCTGGCCGG GQNGNAFAYSKTQQVNSRLCCCCGGCACCTTCTTTGTCTATCAGGTGGTGTTTGTCTATGCGCAC DLYYLSAITQDRTVIVESSAACGCCACTTCGGCCGGTGGGCAGAATGGCAATGCCTTTGCCTATA NAIDPLDWDTVQRNVLIQNGCAAGACCCAGCAGGTGAACTCGCGGCTCGACCTTTACTACCTGTC YNPASNSGHFSFDWSAYNDGGCCATCACCCAGGACCGTACGGTCATCGTCGAGTCCAGCAATGCA PHRRYATCGACCCGCTGGACTGGGATACCGTGCAGCGCAACGTGCTGATCCAGAACTACAACCCGGCCAGCAACAGCGGGCACTTCTCGTTCGACTGGAGCGCCTACAACGATCCTCATCGCCGTTAT ZIP9 SEQ ID NO: 15 SEQ ID NO: 16ATGACGATCAAGGAAGAGCTGAGCCAACCCCAAAGCCATTCGGTCG MTIKEELSQPQSHSVELDQAGCTCGACCAGTTGCAAGTCGGGGAAGTCTCTGCACGCGAAGCGTT LQVGEVSAREALTSNFAGSGACCTCCAACTTCGCCGGCAGTTTCGATCAGTTCCCGACCAAAAGC FDQFPTKSGGFEIDKYLLNGGCGGCTTCGAGATCGACAAATACCTGCTCAACTACGCGGATCCGA YADPKKGCWLDGVTVYGDIAAAAAGGCTGCTGGCTGGACGGCGTCACCGTCTACGGTGACATCTA YIGKQNWGTYTRPVFAYLQCATCGGCAAGCAGAACTGGGGCACCTACACGCGCCCGGTGTTCGCC HTDTISIPQQVTQTKSYQLTACCTGCAGCACACCGACACCATCTCGATTCCGCAGCAGGTGACGC SKGHTQPFTKSVSAKYSVGAGACCAAGAGCTACCAGTTGAGCAAAGGCCACACCCAGCCGTTCAC GSIDIVNISSDITVGFSSTCAAGTCGGTCAGCGCCAAATACAGCGTTGGCGGCAGTATCGACATC EAWSTNQTFTQSTELAGPGGTCAACATCAGCTCGGATATCACCGTTGGTTTCAGCAGCACCGAGG TFFVYQVVFVYAHNATSAGCCTGGTCGACGAACCAGACCTTCACCCAAAGCACCGAGCTGGCCGG GQNGNAFAYSKTQQVNSRLCCCTGGCACCTTCTTTGTCTATCAGGTGGTGTTTGTCTATGCGCAC DLYYLSAITQDRTVIVESSAACGCCACTTCGGCCGGTGGGCAGAATGGCAATGCCTTTGCCTATA NAIDPLDWDTVQRNVLIQNGCAAGACCCAGCAGGTGAACTCGCGGCTCGACCTTTACTACCTGTC YNPASNSGHFSFDWSAYNDGGCCATCACCCAGGACCGTACGGTCATCGTCGAGTCCAGCAATGCA PHRRYATCGACCCGCTGGACTGGGATACCGTGCAGCGCAACGTGCTGATCCAGAACTACAACCCGGCCAGCAACAGCGGGCACTTCTCGTTCGACTGGAGCGCCTACAACGATCCTCATCGCCGTTAT ZIP10 SEQ ID NO: 17 SEQ ID NO: 18ATGACGATCAAGGAAGAGCTGAGCCAACCCCAAAGCCATTCGGTCG MTIKEELSQPQSHSVELDQAGCTCGACCAGTTGCAAGTCGGGGAAGTCTCTGCACGCGAAGCGTT LQVGEVSAREALTSNFAGSGACCTCCAACTTCGCCGGCAGTTTCGATCAGTTCCCGACCAAAAGC FDQFPTKSGSFEIDKYLLNGGCAGCTTCGAGATCGACAAATACCTGCTCAACTACGCGGATCCGA YADPKKGCWLDGVTVYGDIAAAAAGGCTGCTGGCTGGACGGCGTCACCGTCTACGGTGACATCTA YIGKQNWGTYTRPVFAYLQCATCGGCAAGCAGAACTGGGGCACCTACACGCGCCCGGTGTTCGCC HTDTISIPQQVTQTKSYQLTACCTGCAGCACACGGACACCATCTCGATTCCGCAGCAGGTGACGC SKGHTQSFTKSVSAKYSVGAGACCAAGAGCTACCAGTTGAGCAAAGGCCACACCCAGTCGTTCAC GSIDIVNVSSDITVGFSSTCAAGTCGGTCAGCGCCAAGTACAGCGTTGGCGGCAGTATCGACATC EAWSTTQTFTQSTELAGPGGTCAACGTCAGCTCGGATATCACTGTCGGTTTCAGCAGCACCGAGG TFFVYQVVFVYAHNATSAGCCTGGTCGACGACCCAGACCTTCACCCAAAGCACCGAGCTGGCCGG GQNGNAFAYSKTQQVNSRLTCCGGGCACCTTCTTTGTCTATCAGGTGGTGTTTGTCTATGCGCAC DLYYLSAITXDRTVIVESSAACGCCACTTCGGCCGGTGGGCAGAATGGCAATGCCTTTGCCTATA NAIDPLDRDTVQRNVLIQNGCAAGACCCAGCAGGTGAACTCGCGGCTCGACCTTTACTACCTGTC YNPASNSGHFSFDWSAYTIGGCCATCACCCANGACCGTACGGTCATCGTCGAGTCCAGCAATGCA LIAVIELRSGCATCGACCCGCTGGACCGGGATACCGTGCAGCGCAACGTGCTGATCCAGAACTACAACCCGGCCAGCAACAGCGGGCACTTCTCGTTCGACTGGAGCGCCTACACGATCCTCATCGCCGTTATTGAACTGAGATCCGGC TGC ZIP11 SEQ ID NO: 19SEQ ID NO: 20 ATGACGATCAAGGAAGAGCTGAGCCAACCCCAAAGCCATTCGGTCGMTIKEELSNPQSHSVELDQ AGCTCGACCAGTTGCAAGTCGGGGAAGTCTCTGCACGCGAAGCGTTLQVGEVSAREALTANFAGS GACCTCCAACTTCGCCGGCAGTTTCGATCAGTTCCCGACCAAAAGCFDQFPTKSGSFEIDKYLLN GGCAGCTTCGAGATCGACAAATACCTGCTCAACTACGCGGATCCGAYADPKKGCWLDGVTVYGDI AAAAAGGCTGCTGGCTGGACGGCGTCACCGTCTACGGTGACATCTAYIGKQNWGTYTRPVFAYLQ CATCGGCAAGCAGAACTGGGGCACCTACACGCGCCCGGTGTTCGCCHTDTISIPQQVTQTKSYQL TACCTGCAGCACACCGACACCATCTTGATTCCGCAGCAGGTGACGCSKGHTQSFTKSVSAKYSVG AGACCAAGAGCTACCAGTTGAGCAAAGGCCACACCCAGTCGTTCACGSIDIVNISSDITVGFSST CAAGTCGGTCAGCGCCAAATACAGCGTTGGCGGCAGTATCGACATCEAWSTNQTFTQSTELAGPG GTCAACATCAGCTCGGATATCACCGTTGGTTTCAGCAGCACCGAGGTFFVYQVVFVYAHNATSAG CCTGGTCGACGAACCAGACCTTCACCCAAAGCACCGAGCTGGCCGGGQNGNAFAYSKTQQVNSRL CCCCGGCACCTTCTTTGTCTATCAGGTGGTGTTTGTCTATGCGCACDLYYLSAITQDRTVIVESS AACGCCACTTCGGCCGGTGGGCAGAATGGCAATGCCTTTGCCTATANAIDPLDWDTVQRNVLIQN GCAAGACCCAGCAGGTGAACTCGCGGCTCGACCTTTACTACCTGTCYNPASNSGHFSFDWSAYND GGCCATCACCCAGGACCGTACGGTCATCGTCGAGTCCAGCAATGCA PHRRYATCGACCCGCTGGACTGGGATACCGTGCAGCGCAACGTGCTGATCCAGAACTACAACCCGGCCAGCAACAGCGGGCACTTCTCGTTCGACTGGAGCGCCTACAACGATCCTCATCGCCGTTAT ZIP12 SEQ ID NO: 21 SEQ ID NO: 22ATGACGATCAAGGAAGAGCTGGGCCAACCCCAAAGCCATTCGGTCG MTIKEELGQPQSHSVELDQAGCTCGACCAGTTGCAAGTCGGGGAAGTCTCTGCACGCGAAGCGTT LQVGEVSAREALTSNFAGSGACCTCCAACTTCGCCGGCAGTTTCGATCAGTTCCCGACCAAAAGC FDQFPTKSGSFEIDKYLLNGGCAGCTTCGAGATCGACAAATACCTGCTCAACTACGCGGATCCGA YADPKKGCRLDGVTVYGDIAAAAAGGCTGCCGGCTGGACGGCGTCACCGTCTACGGTGACATCTA YIGKQNWGTYTRPVFAYLQCATCGGCAAGCAGAACTGGGGCACCTACACGCGCCCGGTGTTCGCC HTDTISIPQQVTQTRSYQLTACCTGCAGCACACCGACACCATCTCGATTCCGCAGCAGGTGACAC SKGHTQSFTKSVSAKYSVGAGACTCGCAGCTACCAGTTGAGCAAAGGCCACACCCAGTCGTTCAC GSIDIVNISSDITVGFSSTCAAGTCGGTCAGCGCCAAATACAGCGTTGGCGGCAGTATCGACATC EAWSTNQTFTQSTELAGPGGTCAACATCAGCTCGGATATCACCGTTGGTTTCAGCAGCACCGAGG TFFVYQVVFVYAHNATSAGCCTGGTCGACGAACCAGACCTTCACCCAAAGCACCGAGCTGGCCGG GQNGNAFAYSKTQQVNSRLCCCTGGCACCTTCTTTGTCTATCAGGTGGTGTTTGTCTATGCGCAC DLYYLSAITQDRTVIVESSAACGCCACTTCGGCCGGTGGGCAGAATGGCAATGCCTTTGCCTATA NAIDPLDWDTVQRNVLIQNGCAAGACCCAGCAGGTGAACTCGCGGCTCGACCTTTACTACCTGTC YNPASNSGHFSFDWSAYNDGGCCATCACCCAGGACCGTACGGTCATCGTCGAGTCCAGCAATGCA PHRRYATCGACCCGCTGGACTGGGATACCGTGCAGCGCAACGTGCTGATCCAGAACTACAACCCGGCCAGCAACAGCGGGCACTTCTCGTTCGACTGGAGCGCCTACAACGATCCTCATCGCCGTTAT ZIP13 SEQ ID NO: 23 SEQ ID NO: 24ATGACGATCAAGGAAGAGCTGGGCCAACCCCAAAGCCGTTCGGTCG MTIKEELGQPQSRSVELDQAGCTCGACCAGTTGCAAGTCGGGGAAGTCTCTGCACGCGAAGCGTT LQVGEVSAREALTSNFAGSGACCTCCAACTTCGCCGGCAGTTTCGATCAGTTCCCGACCAAAAGC FDQFPTKSGSFEIDKYLLNGGCAGCTTCGAGATCGACAAATACCTGCTCAACTACGCGGATCCGA YADPKKGCWLDGVTVYGDIAAAAAGGCTGCTGGCTGGACGGCGTCACCGTCTACGGTGACATCTA YIGKQNWGTYTRPVFAYLQCATCGGCAAGCAGAACTGGGGCACCTACACGCGCCCGGTGTTCGCC HTDTISIPQQVTQTKSYQLTACCTGCAGCACACCGACACCATCTCGATTCCGCAGCAGGTGACGC SKGHTQSFTKSVSAKYSVGAGACCAAGAGCTACCAGTTGAGCAAAGGCCACACCCAGTCGTTCAC GSIDIVNISSDITVGFSSTCAAGTCGGTCAGCGCCAAATACAGCGTTGGCGGCAGTATCGACATC EAWSTNQTFTQSTELAGPGGTCAACATCAGCTCGGATATCACCGTTGGTTTCAGCAGCACCGAGG TFFVYQVVFVYAHNATSAGCCTGGTCGACGAACCAGACCTTCACCCAAAGCACCGAGCTGGCCGG GQNGNAFAYSKTQQVNSRLCCCTGGCACCTTCTTTGTCTATCAGGTGGTGTTTGTCTATGCGCAC DLYYLSAITQDRTVIVESSAACGCCACCTCGGCCGGTGGGCAGAATGGCAATGCCTTTGCCTATA NAIDPLDWDTVQRNVLIQNGCAAGACCCAGCAGGTGAACTCGCGGCTCGACCTTTACTACCTGTC YNPASNSGHFSFDWSAYNDGGCCATCACCCAGGACCGTACGGTCATCGTCGAGTCCAGCAATGCA PHRRYATCGACCCGCTGGACTGGGATACCGTGCAGCGCAACGTGCTGATCCAGAACTACAACCCGGCCAGCAACAGCGGGCACTTCTCGTTCGACTGGAGCGCCTACAACGATCCTCATCGCCGTTAT ZIP16 SEQ ID NO: 25 SEQ ID NO: 26ATGACGATCAAGGAAGAGCTGGGCCAGCCTCAAAGCCATTCGATCG MTIKEELGQPQSHSIELDEAACTGGACGAGGTGAGCAAGGAGGCCGCAAGTACGCGGGCCGCGTT VSKEAASTRAALTSNLSGRGACTTCCAACCTGTCTGGCCGCTTCGACCAGTACCCGACCAAGAAG FDQYPTKKGDFAIDGYLLDGGCGACTTTGCGATCGATGGTTATTTGCTGGACTACAGCTCACCCA YSSPKQGCWVDGITVYGDIAGCAAGGTTGCTGGGTGGACGGTATCACTGTCTATGGCGATATCTA YIGKQNWGTYTRPVFAYLQCATCGGCAAGCAGAACTGGGGCACTTATACCCGCCCGGTGTTTGCC YVETISIPQNVTTTLSYQLTACCTACAGTATGTGGAAACCATCTCCATTCCACAGAATGTGACGA TKGHTRSFETSVNAKYSVGCCACCCTCAGCTATCAGCTGACCAAGGGGCATACCCGTTCCTTCGA ANIDIVNVGSEISTGFTRSGACCAGTGTCAACGCCAAGTACAGCGTTGGCGCCAACATAGATATC ESWSTTQSFTDTTEMKGPGGTCAACGTGGGTTCGGAGATTTCCACCGGGTTTACCCGCAGCGAGT TFVIYQVVLVYAHNATSAGCCTGGTCCACCACGCAGTCGTTCACCGATACCACCGAGATGAAGGG RQNANAFAYSKTQAVGSRVGCCAGGGACGTTCGTCATTTACCAGGTCGTGCTGGTGTATGCGCAC DLYYLSAITQRKRVIVPSSAACGCCACCTCGGCAGGGCGGCAGAATGCCAATGCCTTCGCCTACA NAVTPLDWDTVQRNVLMENGCAAAACCCAGGCAGTGGGCTCGCGGGTGGACTTGTACTACTTGTC YNPGSNSGHFGFDWSAYNDGGCCATTACCCAGCGCAAGCGGGTCATCGTTCCGTCGAGCAATGCC PHRRYGTCACGCCGCTGGACTGGGATACGGTGCAACGCAACGTGCTGATGGAAAACTACAACCCAGGCAGTAACAGCGGACACTTCGGCTTCGACTGGAGTGCCTACAACGATCCTCATCGCCGTTAT ZIP17 SEQ ID NO: 27 SEQ ID NO: 28ATGACGATCAAGGAAGAGCTGAGCCAACCCCAAAGCCATTCGGTCG MTIKEELSQPQSHSVELDQAGCTCGACCAGTTGCAAGTCGGGGAAGTCTCTGCACGCGAAGCGTT LQVGEVSAREALTSNFAGSGACCTCCAACTTCGCCGGCAGTTTCGATCAGTTCCCGACCAAAGGC FDQFPTKGGSFEIDKYLLNGGCAGCTTCGAGATCGACAAATACCTGCTCAACTACGCGGATCCGA YADPKKGCWLDGVTVYGDIAAAAAGGCTGCTGGCTGGACGGCGTCACCGTCTACGGTGACATCTA YIGKQNWGTYTRPVFAYLQCATCGGCAAGCAGAACTGGGGCACCTACACGCGCCCGGTGTTCGCC HTDTISIPQQVTQTKSYQLTACCTGCAGCACACCGACACCATCTCGATTCCGCAGCAGGTGACGC SKGHTQSFTKPVSAKYSVGAGACCAAGAGCTACCAGTTGAGCAAAGGCCACACCCAGTCGTTCAC GSIDIVNISSDITVGFSSTCAAGCCGGTCAGCGCCAAATACAGCGTTGGCGGCAGTATCGACATC EAWSTNQTFTQSTELAGPGGTCAACATCAGCTCGGATATCACCGTTGGTTTCAGCAGCACCGAGG TFFVYQVVFVYAHNATSAGCCTGGTCGACGAACCAGACCTTCACCCAAAGCACCGAGCTGGCCGG GQNGNAFAYSKTQQVNSRLCCCTGGCACCTTCTTTGTCTATCAGGTGGTGTTTGTCTATGCGCAC DLYYLSAITQDRTVIVESSAACGCCACTTCGGCCGGTGGGCAGAATGGCAATGCCTTTGCCTATA NAIDPLDWDTVQRNVLIQNGCAAGACCCAGCAGGTGAACTCGCGGCTCGACCTTTACTACCTGTC YNPASNSGHFSFDWSAYNDGGCCATCACCCAGGACCGTACGGTCATCGTCGAGTCCAGCAATGCA PHRRYATCGACCCGCTGGACTGGGATACCGTGCAGCGCAACGTGCTGATCCAGAACTACAACCCGGCCAGCAACAGCGGGCACTTCTCGTTCGACTGGAGCGCCTACAACGATCCTCATCGCCGTTAT ZIP18 SEQ ID NO: 29 SEQ ID NO: 30ATGACGATCAAGGAAGAGCTGAGCCAACCCCAAAGCCATTCGGTCG MTIKEELSQPQSHSVELDQAGCTCGACCAGTTGCAAGTCGGGGAAGTCTCTGCACGCGAAGCGTT LQVGEVSAREALTSNFAGSGACCTCCAACTTCGCCGGCAGTTTCGATCAGTTCCCGACCAAAAGC FDQFPTKSGSFEIDKYLLNGGCAGCTTCGAGATCGACAAATACCTGCTCAACTACGCGGATCCGA YADPKKGCWLDGVTVYGDIAAAAAGGCTGCTGGCTGGACGGCGTCACCGTCTACGGTGACATCTA YIGKQNWGTYTRPVFAYLQCATCGGCAAGCAGAACTGGGGCACCTACACGCGCCCGGTGTTCGCC HTDTISIPQQVTQTKSYQLTACCTGCAGCACACCGACACCATCTCGATTCCGCAGCAGGTGACGC SKGHTQSFTKSVSAKYSVGAGACCAAGAGCTACCAGTTGAGCAAAGGCCACACCCAGTCGTTCAC GSIDIVNISSDITVGFSSTCAAGTCGGTCAGCGCCAAATACAGCGTTGGCGGCAGTATCGACATC EAWSTNQTFTQSTELAGPGGTCAACATCAGCTCGGATATCACCGTTGGTTTCAGCAGCACCGAGG TFFVYQVVSVYAHNATSAGCCTGGTCGACGAACCAGACCTTCACCCAAAGCACCGAGCTGGCCGG GQNGNAFAYSKTQQVNSRLCCCTGGCACCTTCTTTGTCTATCAGGTGGTGTCTGTCTATGCGCAC DLYYLSAITQDRTVIVESSAACGCCACTTCGGCCGGTGGGCAGAATGGCAATGCCTTTGCCTATA NAIDPLDWDTVQRNVLIQNGCAAGACCCAGCAGGTGAACTCGCGGCTCGACCTTTACTACCTGTC YNPASNSGHFSFDWSAYNDGGCCATCACCCAGGACCGTACGGTCATCGTCGAGTCCAGCAATGCA PHRRYATCGACCCGCTGGACTGGGATACCGTGCAGCGCAACGTGCTGATCCAGAACTACAACCCGGCCAGCAACAGCGGGCACTTCTCGTTCGACTGGAGCGCCTACAACGATCCTCATCGCCGTTAT ZIP19 SEQ ID NO: 31 SEQ ID NO: 32ATGACGATCAAGGAAGAGCTGAGCCAACCCCAAAGCCATTCGGTCG MTIKEELSQPQSHSVELDQAGCTCGACCAGTTGCAAGTCGGGGAAGTCTCTGCACGCGAAGCGTT LQVGEVSAREALTSNFAGSGACCTCCAACTTCGCCGGCAGTTTCGATCAGTTCCCGACCAAAAGC FDQFPTKSGSFEIDKYLLNGGCAGCTTCGAGATCGACAAATACCTGCTCAACTACGCGGATCCGA YADPKKGCWLDGVTVYGDIAAAAAGGCTGCTGGCTGGACGGCGTCACCGTCTACGGTGACATCTA YIGKQNWGTYTRPVFAYLQCATCGGCAAGCAGAACTGGGGCACCTACACGCGCCCGGTGTTCGCC HTDTISIPQQVTQTKSYQLTACCTGCAGCACACCGACACCATCTCGATTCCGCAGCAGGTGACGC SKGHTQSFTKSVSAKYSVGAGACCAAGAGCTACCAGTTGAGCAAAGGCCACACCCAGTCGTTCAC GGIDIVNISSDITVGFSSTCAAGTCGGTCAGCGCCAAATACAGCGTTGGCGGCGGTATCGACATC EAWSTNQTFTQSTELAGPGGTCAACATCAGCTCGGATATCACCGTTGGTTTCAGCAGCACCGAGG TFFVYQVVFVYAHNATSAGCCTGGTCGACGAACCAGACCTTCACCCAAAGCACCGAGCTGGCCGG GQNGNAFAYSKTQQVNSRLCCCTGGCACCTTCTTTGTCTATCAGGTGGTGTTTGTCTATGCGCAC DLYCLSAITQDRTVIVESSAACGCCACTTCGGCCGGTGGGCAGAATGGCAATGCCTTTGCCTATA NAIDPLDWDTVQRNVLIQNGCAAGACCCAGCAGGTGAACTCGCGGCTCGACCTTTACTGCCTGTC YNPASNSGHFSFDWSAYNDGGCCATCACCCAGGACCGTACGGTCATCGTCGAGTCCAGCAATGCA PHRRYATCGACCCGCTGGACTGGGATACCGTGCAGCGCAACGTGCTGATCCAGAACTACAACCCGGCCAGCAACAGCGGGCACTTCTCGTTCGACTGGAGCGCCTACAACGATCCTCATCGCCGTTAT ZIP20 SEQ ID NO: 33 SEQ ID NO: 34ATGACGATCAAGGAAGAGCTGAGCCAACCCCAAAGCCATTCGGTCG MTIKEELSQPQSHSVELDQAGCTCGACCAGTTGCAAGTCGGGGAAGTCTCTGCACGCGAAGCGTT LQVGEVSAREALTSNFAGSGACCTCCAACTTCGCCGGCAGTTTCGATCAGTTCCCGACCAAAAGC FDQFPTKSGSFEIDKYLLNGGCAGCTTCGAGATCGACAAATACCTGCTCAACTACGCGGATCCGA YADPKKGCWLDGVTVYGDIAAAAAGGCTGCTGGCTGGACGGCGTCACCGTCTACGGTGACATCTA YIGKQNWGTYTRPVFAYLQCATCGGCAAGCAGAACTGGGGCACCTACACGCGCCCGGTGTTCGCC HTDTISIPQQVTQTKSYQLTACCTGCAGCACACCGACACCATCTCGATTCCGCAGCAGGTGACGC SKGHTQSFTKSVSAKYSVGAGACCAAGAGCTACCAGTTGAGCAAAGGCCACACCCAGTCGTTCAC GSIDIVNISSDITVGFSSTCAAGTCGGTCAGCGCCAAATACAGCGTTGGCGGCAGTATCGACATC EAWSTNQTFTQSTELAGPGGTCAACATCAGCTCGGATATCACCGTTGGTTTCAGCAGCACCGAGG TFFVYQVVFVYAHNATSAGCCTGGTCGACGAACCAGACCTTCACCCAAAGCACCGAGCTGGCCGG GQNGNAFAYSKTQQVNSRLCCCTGGCACCTTCTTTGTCTATCAGGTGGTGTTTGTCTATGCGCAC DLYYLSAITQDRTVIVESSAACGCCACTTCGGCCGGTGGGCAGAATGGCAATGCCTTTGCCTATA NAIDPPDWDTVQRNVLIQNGCAAGACCCAGCAGGTGAACTCGCGGCTCGACCTTTACTACCTGTC YNPASNSGHFSFDWSAYNDGGCCATCACCCAGGACCGTACGGTCATCGTCGAGTCCAGCAATGCA PHRRYATCGACCCGCCGGACTGGGATACCGTGCAGCGCAACGTGCTGATCCAGAACTACAACCCGGCCAGCAACAGCGGGCACTTCTCGTTCGACTGGAGCGCCTACAACGATCCTCATCGCCGTTAT ZIP21 SEQ ID NO: 35 SEQ ID NO: 36ATGACGATCAAGGAAGAGCTGAGCCAACCCCAAAGCCATTCGGTCG MTIKEELSQPQSHSVELDQAGCTCGACCAGTTGCAAGTCGGGGAAGTCTCTGCACGCGAAGCGTT LQVGEVSAREALTSNFAGSGACCTCCAACTTCGCCGGCAGTTTCGATCAGTTCCCGACCAAAAGC FDQFPTKSGSFEIDKYLLNGGCAGCTTCGAGATCGACAAATACCTGCTCAACTACGCGGATCCGA YADPKKGCWLDGVTVYGDIAAAAAGGCTGCTGGCTGGACGGCGTCACCGTCTACGGTGACATCTA YIGKQNWGTYTRPVFAYLQCATCGGCAAGCAGAACTGGGGCACCTACACGCGCCCGGTGTTCGCC HTDTISIPQQVTQTKSYQLTACCTGCAGCACACCGACACCATCTCGATTCCGCAGCAGGTGACGC SKGHTQSFTKSVSAKYSVGAGACCAAGAGCTACCAGTTGAGCAAAGGCCACACCCAGTCGTTCAC GSIDIVNISSDITVGFSSTCAAGTCGGTCAGCGCCAAATACAGCGTTGGCGGCAGTATCGACATC EAWSTNQTFTQSTELAGPGGTCAACATCAGCTCGGATATCACCGTTGGTTTCAGCAGCACCGAGG TFFVYQVVFVYAHNATSAGCCTGGTCGACGAACCAGACCTTCACCCAAAGCACCGAGCTGGCCGG GQNGNAFAYGKTQQVNSRLCCCTGGCACCTTCTTTGTCTATCAGGTGGTGTTTGTCTATGCGCAC DLYYLSAITQDRTVIVESSAACGCCACTTCGGCCGGTGGGCAGAATGGCAATGCCTTTGCCTATG NAIDPLDWDTVQRNVLIQNGCAAGACCCAGCAGGTGAACTCGCGGCTCGACCTTTACTACCTGTC YNPASNSGHFSFDWSAYNDGGCCATCACCCAGGACCGTACGGTCATCGTCGAGTCCAGCAATGCA PHRRYATCGACCCGCTGGACTGGGATACCGTGCAGCGCAACGTGCTGATCCAGAACTACAACCCGGCCAGCAACAGCGGGCACTTCTCGTTCGACTGGAGCGCCTACAACGATCCTCATCGCCGTTAT ZIP22 SEQ ID NO: 37 SEQ ID NO: 38ATGACGATCAAGGAAGAGCTGGGCCAACCCCAAAGCCATTCGGTCG MTIKEELGQPQSHSVELDQAGCTCGACCAGTTGCAAGTCGGGGAAGTCTCTGCACGCGAAGCGTT LQVGEVSAREALTSNFAGSGACCTCCAACTTCGCCGGCAGTTTCGATCAGTTCCCGACCAAAAGC FDQFPTKSGSFEIDKYLLNGGCAGCTTCGAGATCGACAAATACCTGCTCAACTACGCGGATCCGA YADPKKGCWLDGVTVYGDIAAAAAGGCTGCTGGCTGGACGGCGTCACCGTCTACGGTGACATCTA YIGKQNWGTYTRPVFAYLQCATCGGCAAGCAGAACTGGGGCACCTACACGCGCCCGGTGTTCGCC HTDTISIPQQVTQTKSYQLTACCTGCAGCACACCGACACCATCTCGATTCCGCAGCAGGTGACGC SKGHTQSFTKSVSAKYSVGAGACCAAGAGCTACCAGTTGAGCAAAGGCCACACCCAGTCGTTCAC GSIDIVNISSDITVGFSSTCAAGTCGGTCAGCGCCAAATACAGCGTTGGCGGCAGTATCGACATC EAWSTNQTFTQSTELAGPGGTCAACATCAGCTCGGATATCACCGTTGGTTTCAGCAGCACCGAGG TFFVYQVVFVYAHNATSAGCCTGGTCGACGAACCAGACCTTCACCCAAAGCACCGAGCTGGCCGG GQNGNAFAYSKTQQVNSRLCCCTGGCACCTTCTTTGTCTATCAGGTGGTGTTTGTCTATGCGCAC DLYYLSAITQDRTVIVESSAACGCCACTTCGGCCGGTGGGCAGAATGGCAATGCCTTTGCCTATA NAIDPLDWDTVQRNVLIQNGCAAGACCCAGCAGGTGAACTCGCGGCTCGACCTTTACTACCTGTC YNPASNSGHFSFDWSAYNDGGCCATCACCCAGGACCGTACGGTCATCGTCGAGTCCAGCAATGCA PHRRYATCGACCCGCTGGACTGGGATACCGTGCAGCGCAACGTGCTGATCCAGAACTACAACCCGGCCAGCAACAGCGGGCACTTCTCGTTCGACTGGAGCGCCTACAACGATCCTCATCGCCGTTAT ZIP23 SEQ ID NO: 39 SEQ ID NO: 40ATGACGATCAAGGAAGAGCTGAGCCAACCCCAAAGCCATTCGGTCG MTIKEELSQPQSHSVELDQAGCTCGACCAGTTGCAAGTCGGGGAAGTCTCTGCACGCGAAGCGTT LQVGEVSAREALTSNFAGSGACCTCCAACTTCGCCGGCAGTTTCGATCAGTTCCCGACCAAAAGC FDQFPTKSGSFEIDKYLLNGGCAGCTTCGAGATCGACAAATACCTGCTCAACTACGCGGATCCGA YADPKKGCWLDGVTVYGDIAAAAAGGCTGCTGGCTGGACGGCGTCACCGTCTACGGTGACATCTA YIGKQNWGTYTRPVFAYLQCATCGGCAAGCAGAACTGGGGCACCTACACGCGCCCGGTGTTCGCC HTDTISIPQQVTQTKSYQSTACCTGCAGCACACCGACACCATCTCGATTCCGCAGCAGGTGACGC SKGHTQSFTKSVSAKYSVGAGACCAAGAGCTACCAGTCGAGCAAAGGCCACACCCAGTCGTTCAC GSIDIVNISSDITVGFSSTCAAGTCGGTCAGCGCCAAATACAGCGTTGGCGGCAGTATCGACATC EAWSTNQTFTQSTELAGPGGTCAACATCAGCTCGGATATCACCGTTGGTTTCAGCAGCACCGAGG TFFVYQVVFVYAHNATSAGCCTGGTCGACGAACCAGACCTTCACCCAAAGCACCGAGCTGGCCGG GQNGNAFAYSKTQQVNSRLCCCTGGCACCTTCTTTGTCTATCAGGTGGTGTTTGTCTATGCGCAC DLYYLSAITQDRTVIVESSAACGCCACTTCGGCCGGTGGGCAGAATGGCAATGCCTTTGCCTATA NAIDPLDWDTVQRNVLIQNGCAAGACCCAGCAGGTGAACTCGCGGCTCGACCTTTACTACCTGTC YNPASNSGHFSFDWSAYNDGGCCATCACCCAGGACCGTACGGTCATCGTCGAGTCCAGCAATGCA PHRRYATCGACCCGCTGGACTGGGATACCGTGCAGCGCAACGTGCTGATCCAGAACTACAACCCGGCCAGCAACAGCGGGCACTTCTCGTTCGACTGGAGCGCCTACAACGATCCTCATCGCCGTTAT ZIP24 SEQ ID NO: 41 SEQ ID NO: 42ATGACGATCAAGGAAGAGCTGAGCCAACCCCAAAGCCATTCGGTCG MTIKEELSQPQSHSVELDQAGCTCGACCAGTTGCAAGTCGGGGAAGTCTCTGCACGCGAAGCGTT LQVGEVSAREALTSNFAGSGACCTCCAACTTCGCCGGCAGTTTCGATCAGTTCCCGACCAAAAGC FDQFPTKSGSFEIDKYLLNGGCAGCTTCGAGATCGACAAATACCTGCTCAACTACGCGGATCCGA YADPKKGCWLDGVTVYGDIAAAAAGGCTGCTGGCTGGACGGCGTCACCGTCTACGGTGACATCTA YIGKQNWGTYTRPVFAYLQCATCGGCAAGCAGAACTGGGGCACCTACACGCGCCCGGTGTTCGCC HTDTISIPQQVTQTKSYQLTACCTGCAGCACACCGACACCATCTCGATTCCGCAGCAGGTGACGC SKGHTQSFTKSVSAKYSVGAGACCAAGAGCTACCAGTTGAGCAAAGGCCACACCCAGTCGTTCAC GSIDIVNISSDITVGFSSTCAAGTCGGTCAGCGCCAAATACAGCGTTGGCGGCAGTATCGACATC EAWSTNQTFTQSTELAGPGGTCAACATCAGCTCGGATATCACCGTTGGTTTCAGCAGCACCGAGG TFFVYQVVFVYAHNATSAGCCTGGTCGACGAACCAGACCTTCACCCAAAGCACCGAGCTGGCCGG GQNGNAFAYSKTQQVNSRLCCCTGGCACCTTCTTTGTCTATCAGGTGGTGTTTGTCTATGCGCAC DLYYLSAITQDRTVIVEPSAACGCCACTTCGGCCGGTGGGCAGAATGGCAATGCCTTTGCCTATA NAIDPLDWDTVQRNVLIQNGCAAGACCCAGCAGGTGAACTCGCGGCTCGACCTTTACTACCTGTC YNPASNSGHFSFDWSAYNDGGCCATCACCCAGGACCGTACGGTCATCGTCGAGCCCAGCAATGCA PHRRYATCGACCCGCTGGACTGGGATACCGTGCAGCGCAACGTGCTGATCCAGAACTACAACCCGGCCAGCAACAGCGGGCACTTCTCGTTCGACTGGAGCGCCTACAACGATCCTCATCGCCGTTAT ZIP25 SEQ ID NO: 43 SEQ ID NO: 44ATGACGATCAAGGAAGAGCTGAGCCAACCCCAAAGCCATTCGGTCG MTIKEELSQPQSHSVELDQAGCTCGACCAGTTGCAAGTCGGGGAAGTCTCTGCACGCGAAGCGTT LQVGEVSAREALTSNFAGSGACCTCCAACTTCGCCGGCAGTTTCGATCAGTTCCCGACCAAAAGC FDQFPTKSGSFEIDKYLLNGGCAGCTTCGAGATCGACAAATACCTGCTCAACTACGCGGATCCGA YADPKKGCWLDGVTVYGDIAAAAAGGCTGCTGGCTGGACGGCGTCACCGTCTACGGTGACATCTA YIGKQNWGTYTRPVFAYLQCATCGGCAAGCAGAACTGGGGCACCTACACGCGCCCGGTGTTCGCC HTDTISIPQQVTQTKSHQLTACCTGCAGCACACCGACACCATCTCGATTCCGCAGCAGGTGACGC SKGHTQSFTKSVSAKYSVGAGACCAAGAGCCACCAGTTGAGCAAAGGCCACACCCAGTCGTTCAC GSIDIVNISSDITVGFSSTCAAGTCGGTCAGCGCCAAATACAGCGTTGGCGGCAGTATCGACATC EAWSTNQTFTQSTELAGPGGTCAACATCAGCTCGGATATCACCGTTGGTTTCAGCAGCACCGAGG TFFVYQVVFVYAHNATSAGCCTGGTCGACGAACCAGACCTTCACCCAAAGCACCGAGCTGGCCGG GQNGNAFAYSKTQQVNSRLCCCTGGCACCTTCTTTGTCTATCAGGTGGTGTTTGTCTATGCGCAC DLYYLSAITQDRTVIVESSAACGCCACTTCGGCCGGTGGGCAGAATGGCAATGCCTTTGCCTATA NAIDPLDWDTVQRNVLIQNGCAAGACCCAGCAGGTGAACTCGCGGCTCGACCTTTACTACCTGTC YNPASNSGHFSFDWSAYNDGGCCATCACCCAGGACCGTACGGTCATCGTCGAGTCCAGCAATGCA PHRRYATCGACCCGCTGGACTGGGATACCGTGCAGCGCAACGTGCTGATCCAGAACTACAACCCGGCCAGCAACAGCGGGCACTTCTCGTTCGACTGGAGCGCCTACAACGATCCTCATCGCCGTTAT ZIP26 SEQ ID NO: 45 SEQ ID NO: 46ATGACGATCAAGGAAGAGCTGAACCAACCCCAAAGCCATTCGGTCG MTIKEELNQPQSHSVELDQAGCTCGACCAGTTGCAAGTCGGGGAAGTCTCTGCACGCGAAGCGTT LQVGEVSAREALTSNFAGSGACCTCCAACTTCGCCGGCAGTTTCGATCAGTTCCCGACCAAAAGC FDQFPTKSGSFEIDKYLLNGGCAGCTTCGAGATCGACAAATACCTGCTCAACTACGCGGATCCGA YADPKKGCWLDGVTVYGDIAAAAAGGCTGCTGGCTGGACGGCGTCACCGTCTACGGTGACATCTA YIGKQNWGTYTRPVFAYLQCATCGGCAAGCAGAACTGGGGCACCTACACGCGCCCGGTGTTCGCC HTDTISIPQQVTQTKSYQLTACCTGCAGCACACCGACACCATCTCGATTCCGCAGCAGGTGACGC SKGHTQSFTKSVSAKYSVGAGACCAAGAGCTACCAGTTGAGCAAAGGCCACACCCAGTCGTTCAC GSIDIVNISSDITVGFSSTCAAGTCGGTCAGCGCCAAATACAGCGTTGGCGGCAGTATCGACATC EAWSTNQTFTQSTELAGPGGTCAACATCAGCTCGGATATCACCGTTGGTTTCAGCAGCACCGAGG TFFVYQVVFVYAHNATSAGCCTGGTCGACGAACCAGACCTTCACCCAAAGCACCGAGCTGGCCGG GQNGNAFAYSKTQQVNSRLCCCTGGCACCTTCTTTGTCTATCAGGTGGTGTTTGTCTATGCGCAC DLYYLSAITQDRTVIVESSAACGCCACTTCGGCCGGTGGGCAGAATGGCAATGCCTTTGCCTATA NAIDPLDWDTVQRNVLIQNGCAAGACCCAGCAGGTGAACTCGCGGCTCGACCTTTACTACCTGTC YNPASNSGHFSFDWSAYNDGGCCATCACCCAGGACCGTACGGTCATCGTCGAGTCCAGCAATGCA PHRRYATCGACCCGCTGGACTGGGATACCGTGCAGCGCAACGTGCTGATCCAGAACTACAACCCGGCCAGCAACAGCGGGCACTTCTCGTTCGACTGGAGCGCCTACAACGATCCTCATCGCCGTTAT ZIP27 SEQ ID NO: 47 SEQ ID NO: 48ATGACGATCAAGGAAGAGCTGAGCCAACCCCAAAGCCATTTGGTCG MTIKEELSQPQSHLVELDQAGCTCGACCAGTTGCAAGTCGGGGAAGTCTCTGCACGCGAAGCGTT LQVGEVSAREALTSNFAGSGACCTCCAACTTCGCCGGCAGTTTCGATCAGTTCCCGACCAAAAGC FDQFPTKSGSFEIDKYLLNGGCAGCTTCGAGATCGACAAATACCTGCTCAACTACGCGGATCCGA YADPKKGCWLDGVTVYGDIAAAAAGGCTGCTGGCTGGACGGCGTCACCGTCTACGGTGACATCTA YIGKQNWGTYTRPVFAYLQCATCGGCAAGCAGAACTGGGGCACCTACACGCGCCCGGTGTTCGCC HTDTISIPQQVTQTKSYQLTACCTGCAGCACACCGACACCATCTCGATTCCGCAGCAGGTGACGC SKGHTQSFTKSVSAKYSVGAGACCAAGAGCTACCAGTTGAGCAAAGGCCACACCCAGTCGTTCAC GSIDIVNISSDITVGFSSTCAAGTCGGTCAGCGCCAAATACAGCGTTGGCGGCAGTATCGACATC EAWSTNQTFTQSTELAGPGGTCAACATCAGCTCGGATATCACCGTTGGTTTCAGCAGCACCGAGG TFFVYQVVFVYAHNATSAGCCTGGTCGACGAACCAGACCTTCACCCAAAGCACCGAGCTGGCCGG GQNGNAFAYSKTQQVNSRLCCCTGGCACCTTCTTTGTCTATCAGGTGGTGTTTGTCTATGCGCAC DLYYLSAITQDRTVIVESSAACGCCACTTCGGCCGGTGGGCAGAATGGCAATGCCTTTGCCTATA NAIDPLDWDTVQRNVLIQNGCAAGACCCAGCAGGTGAACTCGCGGCTCGACCTTTACTACCTGTC YNPASNSGHFSFDWSAYNDGGCCATCACCCAGGACCGTACGGTCATCGTCGAGTCCAGCAATGCA PHRRYATCGACCCGCTGGACTGGGATACCGTGCAGCGCAACGTGCTGATCCAGAACTACAACCCGGCCAGCAACAGCGGGCACTTCTCGTTCGACTGGAGCGCCTACAACGATCCTCATCGCCGTTAT ZIP28 SEQ ID NO: 49 SEQ ID NO: 50ATGACGATCAAGGAAGAGCTGAGCCAACCCCAAAGCCATTCGGTCG MTIKEELSQPQSHSVELDQAGCTCGACCAGTTGCAAGTCGGGGAAGTCTCTGCACGCGAAGCGTT LQVGEVSAREALTSNFAGSGACCTCCAACTTCGCCGGCAGTTTCGATCAGTTCCCGACCAAAAGC FDQFPTKSGSFKIDKYLLNGGCAGCTTCAAGATCGACAAATACCTGCTCAACTACGCGGATCCGA YADPKKGCWLDGVTVYGDIAAAAAGGCTGCTGGCTGGACGGCGTCACCGTCTACGGTGACATCTA YIGKQNWGTYTRPVFAYLQCATCGGCAAGCAGAACTGGGGCACCTACACGCGCCCGGTGTTCGCC HTDTISIPQQVTQTKSYQLTACCTGCAGCACACCGACACCATCTCGATTCCGCAGCAGGTGACGC SKGHTQSFTKSVSAKYSVGAGACCAAGAGCTACCAGTTGAGCAAAGGCCACACCCAGTCGTTCAC GSIDIVNISSDITVGFSSTCAAGTCGGTCAGCGCCAAATACAGCGTTGGCGGCAGTATCGACATC EAWSTNQTFTQSTELAGPGGTCAACATCAGCTCGGATATCACCGTTGGTTTCAGCAGCACCGAGG TFFVYQVVFVYAHNATSAGCCTGGTCGACGAACCAGACCTTCACCCAAAGCACCGAGCTGGCCGG GQNGNAFAYSKTQQVNSRLCCCTGGCACCTTCTTTGTCTATCAGGTGGTGTTTGTCTATGCGCAC DLYYLSAITQDRTVIVESSAACGCCACTTCGGCCGGTGGGCAGAATGGCAATGCCTTTGCCTATA NAIDPLDWDTVQRNVLIQNGCAAGACCCAGCAGGTGAACTCGCGGCTCGACCTTTACTACCTGTC YNPASNSGHFSFDWSAYNDGGCCATCACCCAGGACCGTACGGTCATCGTCGAGTCCAGCAATGCA PHRRYATCGACCCGCTGGACTGGGATACCGTGCAGCGCAACGTGCTGATCCAGAACTACAACCCGGCCAGCAACAGCGGGCACTTCTCGTTCGACTGGAGCGCCTACAACGATCCTCATCGCCGTTAT ZIP29 SEQ ID NO: 51 SEQ ID NO: 52ATGACGATCAAGGAAGAGCTGAGCCAACCCCAAAGCCATTCGGTCG MTIKEELSQPQSHSVELDQAGCTCGACCAGTTGCAAGTCGGGGAAGTCTCTGCACGCGAAGCGTT LQVGEVSAREALTSNFAGSGACCTCCAACTTCGCCGGCAGTTTCGATCAGTTCCCGACCAAAAGC FDQFPTKSGSFEIDKYLLNGGCAGCTTCGAGATCGACAAATACCTGCTCAACTACGCGGATCCGA YADPKKGCWLDGVTVYGDIAAAAAGGCTGCTGGCTGGACGGCGTCACCGTCTACGGTGACATCTA YIGKQNWGTYTRPVFAYLQCATCGGCAAGCAGAACTGGGGCACCTACACGCGCCCGGTGTTCGCC HTDTISIPQQVTQTKSYQLTACCTGCAGCACACCGACACCATCTCGATTCCGCAGCAGGTGACGC SKGHTQSFTKSVSAKYSVGAGACCAAGAGCTACCAGTTGAGCAAAGGCCACACCCAGTCGTTCAC GSIDIVNISSDITVGFSSTCAAGTCGGTCAGCGCCAAATACAGCGTTGGCGGCAGTATCGACATC EAWSTNQTFTRSTELAGPGGTCAACATCAGCTCGGATATCACCGTTGGTTTCAGCAGCACCGAGG TFFVYQVVFVYAHNATSAGCCTGGTCGACGAACCAGACCTTCACCCGAAGCACCGAGCTGGCCGG GQNGNAFAYSKTQQVNSRLCCCTGGCACCTTCTTTGTCTATCAGGTGGTGTTTGTCTATGCGCAC DLYYLSAITQDRTVIVESSAACGCCACTTCGGCCGGTGGGCAGAATGGCAATGCCTTTGCCTATA NAIDPLDWDTVQRNVLIQNGCAAGACCCAGCAGGTGAACTCGCGGCTCGACCTTTACTACCTGTC YNPASNSGHFSFDWSAYNDGGCCATCACCCAGGACCGTACGGTCATCGTCGAGTCCAGCAATGCA PHRRYATCGACCCGCTGGACTGGGATACCGTGCAGCGCAACGTGCTGATCCAGAACTACAACCCGGCCAGCAACAGCGGGCACTTCTCGTTCGACTGGAGCGCCTACAACGATCCTCATCGCCGTTAT ZIP30 SEQ ID NO: 53 SEQ ID NO: 54ATGACGATCAAGGAAGAGCTGAGCCAACCCCAAAGCCATTCGGTCG MTIKEELSQPQSHSVELDQAGCTCGACCAGTTGCAAGTCGGGGAAGTCTCTGCACGCGAAGCGTT LQVGEVSAREALTSNFAGSGACCTCCAACTTCGCCGGCAGTTTCGATCAGTTCCCGACCAAAAGC FDQFPTKSGSFEIDKYLLSGGCAGCTTCGAGATCGACAAATACCTGCTCAGCTACGCGGATCCGA YADPKKGCWLDGVTVYGDIAAAAAGGCTGCTGGCTGGACGGCGTCACCGTCTACGGTGACATCTA YIGKQNWGTYTRPVFAYLQCATCGGCAAGCAGAACTGGGGCACCTACACGCGCCCGGTGTTCGCC HTDTISIPQQVTQTKSYQLTACCTGCAGCACACCGACACCATCTCGATTCCGCAGCAGGTGACGC SKGHTQSFTKSVSAKYSVGAGACCAAGAGCTACCAGTTGAGCAAAGGCCACACCCAGTCGTTCAC GSIDIVNISSDITVGFSSTCAAGTCGGTCAGCGCCAAATACAGCGTTGGCGGCAGTATCGACATC EAWSTNQTFTQSTELAGPGGTCAACATCAGCTCGGATATCACCGTTGGTTTCAGCAGCACCGAGG TFFVYQVVFVYAHNATSAGCCTGGTCGACGAACCAGACCTTCACCCAAAGCACCGAGCTGGCCGG GQNGNAFAYSKTQQVSSRLCCCTGGCACCTTCTTTGTCTATCAGGTGGTGTTTGTCTATGCGCAC DLYYLSAITQDRTVIVESSAACGCCACTTCGGCCGGTGGGCAGAATGGCAATGCCTTTGCCTATA SAIDPLDWDTVQRNVLIQNGCAAGACCCAGCAGGTGAGCTCGCGGCTCGACCTTTACTACCTGTC YNPASNSGHFSFDWSAYNDGGCCATCACCCAGGACCGTACGGTCATCGTCGAGTCCAGCAGTGCA PHRRYATCGACCCGCTGGACTGGGATACCGTGCAGCGCAACGTGCTGATCCAGAACTACAACCCGGCCAGCAACAGCGGGCACTTCTCGTTCGACTGGAGCGCCTACAACGATCCTCATCGCCGTTAT ZIP31 SEQ ID NO: 55 SEQ ID NO: 56ATGACGATCAAGGAAGAGCTGAGCCAACCCCAAAGCCATTCGGTCG MTIKEELSQPQSHSVELDQAGCTCGACCAGTTGCAAGTCGGGGAAGTCTCTGCACGCGAAGCGTT LQVGEVSAREALTSNFAGSGACCTCCAACTTCGCCGGCAGTTTCGATCAGTTCCCGACCAAAAGC FDQFPTKSGSFEIDKYLLNGGCAGCTTCGAGATCGACAAATACCTGCTCAACTACGCGGATCCGA YADPKKGCWLDGVTVYGDIAAAAAGGCTGCTGGCTGGACGGCGTCACCGTCTACGGTGACATCTA YIGKQNWGTYTRPVFAYLQCATCGGCAAGCAGAACTGGGGCACCTACACGCGCCCGGTGTTCGCC HTDTISIPQQVTQTKSYQLTACCTGCAGCACACCGACACCATCTCGATTCCGCAGCAGGTGACGC SKGHTQSFTKSVSAKYSVGAGACCAAGAGCTACCAGTTGAGCAAAGGCCACACCCAGTCGTTCAC GSIDIVNISSDITVGFSSTCAAGTCGGTCAGCGCCAAATACAGCGTTGGCGGCAGTATCGACATC EAWSTNQTFTQSTELAGPGGTCAACATCAGCTCGGATATCACCGTTGGTTTCAGCAGCACCGAGG TFFVYQVVFVYAHNATSAGCCTGGTCGACGAACCAGACCTTCACCCAAAGCACCGAGCTGGCCGG GQNGNAFAYSKTQRVNSRLCCCTGGCACCTTCTTTGTCTATCAGGTGGTGTTTGTCTATGCGCAC DLYYLSAITQDRTVIVESSAACGCCACTTCGGCCGGTGGGCAGAATGGCAATGCCTTTGCCTATA NAIDPLDWDTVQRNVLIQNGCAAGACCCAGCGGGTGAACTCGCGGCTCGACCTTTACTACCTGTC YNPASNSGHFSFDWSAYNDGGCCATCACCCAGGACCGTACGGTCATCGTCGAGTCCAGCAATGCA PHRRYATCGACCCGCTGGACTGGGATACCGTGCAGCGCAACGTGCTGATCCAGAACTACAACCCGGCCAGCAACAGCGGGCACTTCTCGTTCGACTGGAGCGCCTACAACGATCCTCATCGCCGTTAT ZIP32 SEQ ID NO: 57 SEQ ID NO: 58ATGACGATCAAGGAAGAGCTGAGCCAACCCCAAAGCCATTCGGTCG MTIKEELSQPQSHSVELDQAGCTCGACCAGTTGCAAGTCGGGGAAGTCTCTGCACGCGAAGCGTT LQVGEVSAREALTSNFAGSGACCTCCAACTTCGCCGGCAGTTTCGATCAGTTCCCGACCAAAAGC FDQFPTKSGSFEIDKYLLNGGCAGCTTCGAGATCGACAAATACCTGCTCAACTACGCGGATCCGA YADPKKGCWLDGVTVYGDIAAAAAGGCTGCTGGCTGGACGGCGTCACCGTCTACGGTGACATCTA YIGKQNWGTYTRPVFAYLQCATCGGCAAGCAGAACTGGGGCACCTACACGCGCCCGGTGTTCGCC HTDTISIPQQVTQTKSYQLTACCTGCAGCACACCGACACCATCTCGATTCCGCAGCAGGTGACGC SKGHAQSFTKSVSAKYSVGAGACCAAGAGCTACCAGTTGAGCAAAGGCCACGCCCAGTCGTTCAC GSIDIVNISSDITVGFSSTCAAGTCGGTCAGCGCCAAATACAGCGTTGGCGGCAGTATCGACATC EAWSTNQTFTQSTELAGPGGTCAACATCAGCTCGGATATCACCGTTGGTTTCAGCAGCACCGAGG TFFVYQVVFVYAHNATSAGCCTGGTCGACGAACCAGACCTTCACCCAAAGCACCGAGCTGGCCGG GQNGNAFAYSKTQQVNSRLCCCTGGCACCTTCTTTGTCTATCAGGTGGTGTTTGTCTATGCGCAC DLYYLSAITQDRTVIVESSAACGCCACTTCGGCCGGTGGGCAGAATGGCAATGCCTTTGCCTATA NAIDPLDWDTVQRNVLIQNGCAAGACCCAGCAGGTGAACTCGCGGCTCGACCTTTACTACCTGTC YNPASNSGHFSFDWSAYNDGGCCATCACCCAGGACCGTACGGTCATCGTCGAGTCCAGCAATGCA PHRRYATCGACCCGCTGGACTGGGATACCGTGCAGCGCAACGTGCTGATCCAGAACTACAACCCGGCCAGCAACAGCGGGCACTTCTCGTTCGACTGGAGCGCCTACAACGATCCTCATCGCCGTTAT ZIP33 SEQ ID NO: 59 SEQ ID NO: 60ATGACGATCAAGGAAGAGCTGAGCCAACCCCAAAGCCATTCGGTCG MTIKEELSQPQSHSVELDQAGCTCGACCAGTTGCAAGTCGGGGAAGTCTCTGCACGCGAAGCGTT LQVGEVSAREALTSNFAGSGACCTCCAACTTCGCCGGCAGTTTCGATCAGTTCCCGACCAAAAGC FDQFPTKSGNFEIDKYLLNGGCAACTTCGAGATCGACAAATACCTGCTCAACTACGCGGATCCGA YADPKKGCWLDGVTVYGDIAAAAAGGCTGCTGGCTGGACGGCGTCACCGTCTACGGTGACATCTA YIGKQNWGTYTRPVFAYLQCATCGGCAAGCAGAACTGGGGCACCTACACGCGCCCGGTGTTCGCC HTDTISIPQQVTQTKSYQLTACCTGCAGCACACCGACACCATCTCGATTCCGCAGCAGGTGACGC SKGHTQSFTKSVSAKYSVGAGACCAAGAGCTACCAGTTGAGCAAAGGCCACACCCAGTCGTTCAC GSIDIVNISSDITVGFSSTCAAGTCGGTCAGCGCCAAATACAGCGTTGGCGGCAGTATCGACATC EAWSTNQTFTQSTELAGPGGTCAACATCAGCTCGGATATCACCGTTGGTTTCAGCAGCACCGAGG TFFVYQVVFVYAHNATSAGCCTGGTCGACGAACCAGACCTTCACCCAAAGCACCGAGCTGGCCGG GQNGNAFAYSKTQQVNSRLCCCTGGCACCTTCTTTGTCTATCAGGTGGTGTTTGTCTATGCGCAC DLYYLSAITQDRTVIVESSAACGCCACTTCGGCCGGTGGGCAGAATGGCAATGCCTTTGCCTATA NAIDPLDWDTVQRNVLIQNGCAAGACCCAGCAGGTGAACTCGCGGCTCGACCTTTACTACCTGTC YNPASNSGHFSFDWSAYNDGGCCATCACCCAGGACCGTACGGTCATCGTCGAGTCCAGCAATGCA PHRRYATCGACCCGCTGGACTGGGATACCGTGCAGCGCAACGTGCTGATCCAGAACTACAACCCGGCCAGCAACAGCGGGCACTTCTCGTTCGACTGGAGCGCCTACAACGATCCTCATCGCCGTTAT ZIP34 SEQ ID NO: 61 SEQ ID NO: 62ATGACGATCAAGGAAGAGCTGAGCCAACCCCAAAGCCATTCGGTCG MTIKEELSQPQSHSVELDQAGCTCGACCAGTTGCAAGTCGGGGAAGTCTCTGCACGCGAAGCGTT LQVGEVSAREALTSNFAGSGACCTCCAACTTCGCCGGCAGTTTCGATCAGTTCCCGACCAAAAGC FDQFPTKSGSFEIDKYLINGGCAGCTTCGAGATCGACAAATACCTGATCAACTACGCGGATCCGA YADPKKGCWLDGVTVYGDIAAAAAGGCTGCTGGCTGGACGGCGTCACCGTCTACGGTGACATCTA YIGKQNWGTYTRPVFAYLQCATCGGCAAGCAGAACTGGGGCACCTACACGCGCCCGGTGTTCGCC HTDTISIPQQVTQTKSYQLTACCTGCAGCACACCGACACCATCTCGATTCCGCAGCAGGTGACGC SKGHTQSFTKSVSAKYSVGAGACCAAGAGCTACCAGTTGAGCAAAGGCCACACCCAGTCGTTCAC GSIDIVNISSDITVGFSSTCAAGTCGGTCAGCGCCAAATACAGCGTTGGCGGCAGTATCGACATC EAWSTNQTFTQSTELAGPGGTCAACATCAGCTCGGATATCACCGTTGGTTTCAGCAGCACCGAGG TFFVYQVVFVYAHNATSAGCCTGGTCGACGAACCAGACCTTCACCCAAAGCACCGAGCTGGCCGG GQNGNAFAYSKTQQVNSRLCCCTGGCACCTTCTTTGTCTATCAGGTGGTGTTTGTCTATGCGCAC DLYYLSAITQDRTVIVESSAACGCCACTTCGGCCGGTGGGCAGAATGGCAATGCCTTTGCCTATA NAIDPLDWDTVQRNVLIQNGCAAGACCCAGCAGGTGAACTCGCGGCTCGACCTTTACTACCTGTC YNPASNSGHFSFDWSAYNDGGCCATCACCCAGGACCGTACGGTCATCGTCGAGTCCAGCAATGCA PHRRYATCGACCCGCTGGACTGGGATACCGTGCAGCGCAACGTGCTGATCCAGAACTACAACCCGGCCAGCAACAGCGGGCACTTCTCGTTCGACTGGAGCGCCTACAACGATCCTCATCGCCGTTAT ZIP35 SEQ ID NO: 63 SEQ ID NO: 64ATGACGATCAAGGAAGAGCTGAGCCAACCCCAAAGCCATTCGGTCG MTIKEELSQPQSHSVELDQAGCTCGACCAGTTGCAAGTCGGGGAAGTCTCTGCACGCGAAGCGTT LQVGEVSAREALTSNFAGSGACCTCCAACTTCGCCGGCAGTTTCGATCAGTTCCCGACCAAAAGC FDQFPTKSGSFEIDKYLLNGGCAGCTTCGAGATCGACAAATACCTGCTCAACTACGCGGATCCGA YADPKKGCWLDGVTVYGDIAAAAAGGCTGCTGGCTGGACGGCGTCACCGTCTACGGTGACATCTA YIGKQNWGTYTRPVFAYLQCATCGGCAAGCAGAACTGGGGCACCTACACGCGCCCGGTGTTCGCC HTDTISIPQQVTQTKSYQLTACCTGCAGCACACCGACACCATCTCGATTCCGCAGCAGGTGACGC SKGHTQSFTKSVSAKYSVGAGACCAAGAGCTACCAGTTGAGCAAAGGCCACACCCAGTCGTTCAC GSIDIVNISSDITVGFSSTCAAGTCGGTCAGCGCCAAATACAGCGTTGGCGGCAGTATCGACATC EAWSTNQTFTQSTELAGPGGTCAACATCAGCTCGGATATCACCGTTGGTTTCAGCAGCACCGAGG TFFVYQVVFVYAHNATSAGCCTGGTCGACGAACCAGACCTTCACCCAAAGCACCGAGCTGGCCGG GQNGNAFAYSKTQQVNSRLCCCTGGCACCTTCTTTGTCTATCAGGTGGTGTTTGTCTATGCGCAC DLYYLSAITQDRTVIVESSAACGCCACTTCGGCCGGTGGGCAGAATGGCAATGCCTTTGCCTATA NAIDPLDWDTVQRNVLIQNGCAAGACCCAGCAGGTGAACTCGCGGCTCGACCTTTACTACCTGTC YNPASNSGHFSFDWGAYNDGGCCATCACCCAGGACCGTACGGTCATCGTCGAGTCCAGCAATGCA PHRRYATCGACCCGCTGGACTGGGATACCGTGCAGCGCAACGTGCTGATCCAGAACTACAACCCGGCCAGCAACAGCGGGCACTTCTCGTTCGACTGGGGCGCCTACAACGATCCTCATCGCCGTTAT ZIP36 SEQ ID NO: 65 SEQ ID NO: 66ATGACGATCAAGGAAGAGCTGGGCCAGCCTCAAAGCCATTCGATCG MTIKEELGQPQSHSIELDEAACTGGACGAGGTGAGCAAGGAGGCCGCAAGTACGCGGGCCGCGTT VSKEAASTRAALTSNLSGRGACTTCCAACCTGTCTGGCCGCTTCGACCAGTACCCGACCAAGAAG FDQYPTKKGDFAIDGYLLDGGCGACTTTGCGATCGATGGTTATTTGCTGGACTACAGCTCACCCA YSSPKQGCWVDGITVYGDIAGCAAGGTTGCTGGGTGGACGGTATCACTGTCTATGGCGATATCTA YIGKQNWGTYTRPVFAYLQCATCGGCAAGCAGAACTGGGGCACTTATACCCGCCCGGTGTTTGCC YVETISIPQNVTTTLSYQLTACCTACAGTATGTGGAAACCATCTCCATTCCACAGAATGTGACGA TKGHTRSFETSVNAKYSVGCCACCCTCAGCTATCAGCTGACCAAGGGGCATACCCGTTCCTTCGA ANIDIVNVGSEISTGFTRSGACCAGTGTCAACGCCAAGTACAGCGTTGGCGCCAACATAGATATC ESWSTTQSFTDTTEMKGPGGTCAACGTGGGTTCGGAGATTTCCACCGGGTTTACCCGCAGCGAGT TFVIYQVVLVYAHNATSAGCCTGGTCCACCACGCAGTCGTTCACCGATACCACCGAGATGAAGGG RQNANAFAYSKTQAVGSRVGCCAGGGACGTTCGTCATTTACCAGGTCGTGCTGGTGTATGCGCAC DLYYLSAITQRKRVIVPSSAACGCCACCTCGGCAGGGCGGCAGAATGCCAATGCCTTCGCCTACA NAVTPLDWDTVQRNVLMENGCAAAACCCAGGCAGTGGGCTCGCGGGTGGACTTGTACTACTTGTC YNPGSNSGHFSFDWSAYNDGGCCATTACCCAGCGCAAGCGGGTCATCGTTCCGTCGAGCAATGCC PHRRYGTCACGCCGCTGGACTGGGATACGGTGCAACGCAACGTGCTGATGGAAAACTACAACCCAGGCAGTAACAGCGGACACTTCAGCTTCGACTGGAGTGCCTACAACGATCCTCATCGCCGTTAT ZIP37 SEQ ID NO: 67 SEQ ID NO: 68ATGACGATCAAGGAAGAGCTGGGCCAGCCTCAAAGCCATTCGATCG MTIKEELGQPQSHSIELDEAACTGGACGAGGTGAGCAAGGAGGCCGCAAGTACGCGGGCCGCGTT VSKEAASTRAALTSNLSGRGACTTCCAACCTGTCTGGCCGCTTCGACCAGTACCCGACCAAGAAG FDQYPTKKGDFAIDGYLLDGGCGACTTTGCGATCGATGGTTATTTGCTGGACTACAGCTCACCCA YSSPKQGCWVDGITVYGDIAGCAAGGTTGCTGGGTGGACGGTATCACTGTCTATGGCGATATCTA YIGKQNWGTYTRPVFAYLQCATCGGCAAGCAGAACTGGGGCACTTATACCCGCCCGGTGTTTGCC YVETISIPQNVTTTLSYQLTACCTACAGTATGTGGAAACCATCTCCATTCCACAGAATGTGACGA TKGHTRSFETSVNAKYSVGCCACCCTCAGCTATCAGCTGACCAAGGGGCATACCCGTTCCTTCGA ANIDIVNVGSEISTGFTRSGACCAGTGTCAACGCCAAGTACAGCGTTGGCGCCAACATAGATATC ESWSTTQSFTDTTEMKGPGGTCAACGTGGGTTCGGAGATTTCCACCGGGTTTACCCGCAGCGAGT TFVIYQVVLVYAHNATSAGCCTGGTCCACCACGCAGTCGTTCACCGATACCACCGAGATGAAGGG RQNANAFAYSKTQAVGSRVGCCAGGGACGTTCGTCATTTACCAGGTCGTGCTGGTGTATGCGCAC DLYYLSAITQRKRVIVPSSAACGCCACCTCGGCAGGGCGGCAGAATGCCAATGCCTTCGCCTACA NAVTPLDWDTVQRNVLMENGCAAAACCCAGGCAGTGGGCTCGCGGGTGGACTTGTACTACTTGTC YNPGSNSGHFRSDWSAYNDGGCCATTACCCAGCGCAAGCGGGTCATCGTTCCGTCGAGCAATGCC PHRRYGTCACGCCGCTGGACTGGGATACGGTGCAACGCAACGTGCTGATGGAAAACTACAACCCAGGCAGTAACAGCGGACACTTCCGCTCCGACTGGAGCGCCTACAACGATCCTCATCGCCGTTAT ZIP38 SEQ ID NO: 69 SEQ ID NO: 70ATGACGATCAAGGAAGAGCTGAGCCAACCCCAAAGCCATTCGGTCG MTIKEELSQPQSHSVELDQAGCTCGACCAGTTGCAAGTCGGGGAAGTCTCTGCACGCGAAGCGTT LQVGEVSAREALTSNFAGSGACCTCCAACTTCGCCGGCAGTTTCGATCAGTTCCCGACCAAAAGC FDQFPTKSGSFEIDKYLLNGGCAGCTTCGAGATCGACAAATACCTGCTCAACTACGCGGATCCGA YADPKKGCWLDGVTVYGDIAAAAAGGCTGCTGGCTGGACGGCGTCACCGTCTACGGTGACATCTA YIGKQNWGTYTRPVFAYLQCATCGGCAAGCAGAACTGGGGCACCTACACGCGCCCGGTGTTCGCC HTGTISIPQQVTQTKSYQLTACCTGCAGCACACCGGCACCATCTCGATTCCGCAGCAGGTGACGC SKGHTQSFTKSVSAKYSVGAGACCAAGAGCTACCAGTTGAGCAAAGGCCACACCCAGTCGTTCAC GSIDIVNISSDITVGFSSTCAAGTCGGTCAGCGCCAAATACAGCGTTGGCGGCAGTATCGACATC EAWSTNQTFTQSTELAGPGGTCAACATCAGCTCGGATATCACCGTTGGTTTCAGCAGCACCGAGG TFFVYQVVFVYAHNATSAGCCTGGTCGACGAACCAGACCTTCACCCAAAGCACCGAGCTGGCCGG GQNGNAFAYSKTQQVNSRLCCCTGGCACCTTCTTTGTCTATCAGGTGGTGTTTGTCTATGCGCAC DLYYLSAITQDRTVIVESSAACGCCACTTCGGCCGGTGGGCAGAATGGCAATGCCTTTGCCTATA NAIDPLDWDTVQRNVLIQNGCAAGACCCAGCAGGTGAACTCGCGGCTCGACCTTTACTACCTGTC YNPASNSGHFSFDWSAYNDGGCCATCACCCAGGACCGTACGGTCATCGTCGAGTCCAGCAATGCA PHRRYATCGACCCGCTGGACTGGGATACCGTGCAGCGCAACGTGCTGATCCAGAACTACAACCCGGCCAGCAACAGCGGGCACTTCTCGTTCGACTGGAGCGCCTACAACGATCCTCATCGCCGTTAT ZIP39 SEQ ID NO: 71 SEQ ID NO: 72ATGACGATCAAGGAAGAGCTGAGCAATCCTCAAAGCCATTCGGTCG MTIKEELSNPQSHSVELDQAGCTCGACCAGTTGCAAGTCGGGGAAGTCTCTGCACGCGAAGCGTT LQVGEVSAREALTANFAGSGACCGCCAACTTCGCCGGCAGTTTCGATCAGTTCCCGACCAAAAGC FDQFPTKSGSFEIDKYLLNGGCAGCTTCGAGATCGACAAATACCTGCTCAACTACGCAGACCCGA YADPKQGCWLDGVTVYGDIAACAAGGCTGCTGGCTGGACGGCGTCACCGTCTACGGTGACATCTA YIGKQNWGTYTRPVFAYLQCATCGGCAAGCAGAACTGGGGCACCTACACGCGCCCGGTGTTCGCC HTDTISIPQQVTQTKSYQLTACCTGCAGCACACGGACACCATCTCGATTCCGCAGCAGGTGACGC SKGHTQSFTKSVSAKYSVGAGACCAAGAGCTACCAGCTGAGCAAAGGCCACACCCAGTCGTTCAC GSIDIVNVSSDITVGFSSTCAAGTCGGTCAGCGCCAAGTACAGCGTTGGCGGCAGTATCGACATC EAWSTTQTFTQSTELAGPGGTCAACGTCAGCTCGGATATCACTGTCGGTTTCAGCAGCACCGAGG TFFVYQVVFVYAHNATSAGCCTGGTCGACGACCCAGACCTTCACCCAAAGCACCGAGCTGGCCGG RQNGNAFAYSKTQQVDSRLTCCGGGCACCTTCTTTGTCTATCAGGTGGTGTTTGTCTACGCGCAC DLYYLSAITQDRTVIVESSAACGCCACCTCGGCGGGCCGGCAGAATGGCAATGCCTTTGCCTATA KAINPLDWDTVQRNVLIENGCAAGACCCAGCAGGTGGATTCGCGGCTCGATCTCTACTACCTGTC YNPASNSGHFRFDWSAYNDGGCCATCACCCAGGACCGTACGGTCATCGTCGAGTCCAGCAAGGCA PHRRYATCAACCCGCTGGACTGGGATACCGTGCAGCGCAACGTGCTGATCGAGAACTACAACCCGGCCTCCAACAGTGGGCACTTCCGCTTCGACTGGAGCGCCTACAACGATCCTCATCGTCGTTAC

Further, reference can be had to FIG. 4, which illustrates a multiplesequence alignment comparing eight of the discovered insecticidalproteins (i.e., ZIP1, ZIP2, ZIP6, ZIP8, ZIP9, ZIP10, ZIP11, and ZIP12)to that of Monalysin, with a corresponding phylogenetic tree found inFIG. 6.

The below Table 4 is an identity matrix, which illustrates the percentidentity amongst the 32 aforementioned proteins having at least 20%sequence identity difference from any known protein in this class.

Furthermore, Table 5 compares the identity from these newly discovered32 proteins to that of Monalysin, which was the first protein discoveredin this class. As can be seen from Table 5, the taught insecticidalproteins are sufficiently different from Monalysin at the amino acidlevel.

This result further illustrates the power of the taught IPDP to discovernovel proteins.

TABLE 4 Sequence Identity Matrix of 32 Novel Insecticidal Proteins FromTable 3 Identified via the IPDP ZIP1 ZIP2 ZIP3 ZIP5 ZIP6 ZIP8 ZIP9 ZIP10ZIP11 ZIP12 ZIP13 ZIP1 97.42 95.94 95.20 97.05 99.63 99.26 95.96 99.2698.89 99.26 ZIP2 97.42 93.36 92.62 94.47 97.05 96.68 98.57 96.68 96.3196.68 ZIP3 95.94 93.36 94.10 95.20 95.57 95.20 91.90 95.20 94.83 95.20ZIP5 95.20 92.62 94.10 94.10 94.83 94.47 93.38 95.94 94.10 94.47 ZIP697.05 94.47 95.20 94.10 96.68 96.31 93.01 96.31 96.68 97.05 ZIP8 99.6397.05 95.57 94.83 96.68 98.89 95.59 98.89 98.52 98.89 ZIP9 99.26 96.6895.20 94.47 96.31 98.89 95.22 98.52 98.16 98.52 ZIP10 95.96 98.57 91.9093.38 93.01 95.59 95.22 95.22 94.85 95.22 ZIP11 99.26 96.68 95.20 95.9496.31 98.89 98.52 95.22 98.16 98.52 ZIP12 98.89 96.31 94.83 94.10 96.6898.52 98.16 94.85 98.16 98.89 ZIP13 99.26 96.68 95.20 94.47 97.05 98.8998.52 95.22 98.52 98.89 ZIP17 99.26 96.68 95.20 94.47 96.31 98.89 98.5295.22 98.52 98.16 98.52 ZIP18 99.63 97.05 95.94 94.83 96.68 99.26 98.8995.59 98.89 98.52 98.89 ZIP19 99.26 96.68 95.20 94.47 96.31 98.89 98.5295.22 98.52 98.16 98.52 ZIP20 99.63 97.05 95.57 94.83 96.68 99.26 98.8995.59 98.89 98.52 98.89 ZIP21 99.63 97.05 95.94 94.83 95.68 99.26 98.8995.59 98.89 98.52 98.89 ZIP22 99.63 97.05 95.57 94.83 97.42 99.26 98.8995.59 98.89 99.26 99.63 ZIP23 99.63 97.05 95.57 94.83 96.68 99.26 98.8995.59 98.83 98.52 98.89 ZIP24 99.63 97.05 95.57 94.83 96.68 99.26 98.8995.59 98.89 98.52 98.89 ZIP25 99.63 97.05 95.57 94.83 96.68 99.26 98.8995.59 98.89 98.52 98.89 ZIP26 99.63 97.05 95.57 94.83 97.05 99.26 98.8995.59 98.89 98.89 99.26 ZIP27 99.63 97.05 95.57 94.83 96.68 99.26 98.8995.59 98.89 98.52 98.89 ZIP28 99.63 97.05 95.57 94.83 96.68 99.26 98.8995.59 98.89 98.52 98.89 ZIP29 99.63 97.05 95.57 94.83 96.68 99.26 98.8995.59 98.89 98.52 98.89 ZIP30 98.89 96.31 95.57 94.83 96.31 98.52 98.1694.85 98.16 97.79 98.16 ZIP31 99.63 97.05 95.57 94.83 96.68 99.26 98.8995.59 98.89 98.52 98.89 ZIP32 99.63 97.05 95.57 94.83 96.68 99.26 98.8995.59 98.89 98.52 98.89 ZIP33 99.63 97.05 95.57 94.83 96.68 99.26 99.2695.59 98.89 98.52 98.89 ZIP34 99.63 97.05 95.57 94.83 96.68 99.26 98.8995.59 98.89 98.52 98.89 ZIP35 99.63 97.05 95.57 94.83 96.68 99.26 98.8995.59 98.89 98.52 98.89 ZIP38 99.63 97.05 95.57 94.83 96.68 99.26 98.8995.59 98.89 98.52 98.89 ZIP39 95.94 93.36 94.47 99.26 94.83 95.57 95.2093.38 96.68 94.83 95.20 ZIP17 ZIP18 ZIP19 ZIP20 ZIP21 ZIP22 ZIP23 ZIP24ZIP25 ZIP26 ZIP27 ZIP1 99.26 99.63 99.26 99.63 99.63 99.63 99.63 99.6399.63 99.63 99.63 ZIP2 96.68 97.05 96.68 97.05 97.05 97.05 97.05 97.0597.05 97.05 97.05 ZIP3 95.20 95.94 95.20 95.57 95.94 95.57 95.57 95.5795.57 95.57 95.57 ZIP5 94.47 94.83 94.47 94.83 94.83 94.83 94.83 94.8394.83 94.83 94.83 ZIP6 96.31 96.68 96.31 96.68 96.68 97.42 96.68 96.6896.68 97.05 96.68 ZIP8 98.89 99.26 98.89 99.26 99.26 99.26 99.26 99.2699.26 99.26 99.26 ZIP9 98.52 98.89 98.52 98.89 98.89 98.89 98.83 98.8998.89 98.89 98.89 ZIP10 95.22 95.59 95.22 95.59 95.59 95.59 95.59 95.5995.59 95.59 95.59 ZIP11 98.52 98.89 98.52 98.89 98.89 98.89 98.89 98.8998.89 98.89 98.89 ZIP12 98.16 98.52 98.16 98.52 98.52 99.26 98.52 98.5298.52 98.89 98.52 ZIP13 98.52 98.89 98.52 98.89 98.89 99.63 98.89 98.8998.89 99.26 98.89 ZIP17 98.89 98.52 98.89 98.89 98.89 98.89 98.89 98.8998.89 98.89 ZIP18 98.89 98.89 99.26 99.26 99.26 99.26 99.26 99.26 99.2699.26 ZIP19 98.52 98.89 98.89 98.89 98.89 98.89 98.89 98.89 98.89 98.89ZIP20 98.89 99.26 98.89 99.26 99.26 99.26 99.26 99.26 99.26 99.26 ZIP2198.89 99.26 98.89 99.26 99.26 99.26 99.26 99.26 99.26 99.26 ZIP22 98.8999.26 98.89 99.26 99.26 99.26 99.26 99.26 99.63 99.26 ZIP23 98.89 99.2698.89 99.26 99.26 99.26 99.26 99.26 99.26 99.26 ZIP24 98.89 99.26 98.8999.26 99.26 99.26 99.26 99.26 99.26 99.26 ZIP25 98.89 99.26 98.89 99.2699.26 99.26 99.26 99.26 99.26 99.26 ZIP26 98.89 99.26 98.89 99.26 99.2699.63 99.26 99.26 99.26 99.26 ZIP27 98.89 99.26 98.89 99.26 99.26 99.2699.26 99.26 99.26 99.26 ZIP28 98.89 99.26 98.89 99.26 99.26 99.26 99.2699.26 99.26 99.26 99.26 ZIP29 98.89 99.26 98.89 99.26 99.26 99.26 99.2699.26 99.26 99.26 99.26 ZIP30 98.16 98.52 98.16 98.52 98.52 98.52 98.5298.52 98.52 98.52 98.52 ZIP31 98.89 99.26 98.89 99.26 99.26 99.26 99.2699.26 99.26 99.26 99.26 ZIP32 98.89 99.26 98.89 99.26 99.26 99.26 99.2699.26 99.26 99.26 99.26 ZIP33 98.89 99.26 98.89 99.26 99.26 99.26 99.2699.26 99.26 99.26 99.26 ZIP34 98.89 99.26 98.89 99.26 99.26 99.26 99.2699.26 99.26 99.26 99.26 ZIP35 98.89 99.26 98.89 99.26 99.26 99.26 99.2699.26 99.26 99.26 99.26 ZIP38 98.89 99.26 98.89 99.26 99.26 99.26 99.2699.26 99.26 99.26 99.26 ZIP39 95.20 95.57 95.20 95.57 95.57 95.57 95.5795.57 95.57 95.57 95.57 ZIP28 ZIP29 ZIP30 ZIP31 ZIP32 ZIP33 ZIP34 ZIP35ZIP38 ZIP39 ZIP1 99.63 99.63 98.89 99.63 99.63 99.63 99.63 99.63 99.6395.94 ZIP2 97.05 97.05 96.31 97.05 97.05 97.05 97.05 97.05 97.05 93.36ZIP3 95.57 95.57 95.57 95.57 95.57 95.57 95.57 95.57 95.57 94.47 ZIP594.83 94.83 94.83 94.83 94.83 94.83 94.83 94.83 94.83 99.25 ZIP6 96.6896.68 96.31 96.68 96.68 96.68 96.68 96.68 96.68 94.83 ZIP8 99.26 99.2698.52 99.26 99.26 99.26 99.26 99.26 99.26 95.57 ZIP9 98.89 98.89 98.1698.89 98.89 99.26 98.89 98.89 98.89 95.20 ZIP10 95.59 95.59 94.85 95.5995.59 95.59 95.59 95.59 95.59 93.38 ZIP11 98.89 98.89 98.16 98.89 98.8998.89 98.89 98.89 98.89 96.63 ZIP12 98.52 98.52 97.79 98.52 98.52 98.5298.52 98.52 98.52 94.83 ZIP13 98.89 98.89 98.16 98.89 98.89 98.89 98.8998.89 98.89 95.20 ZIP17 98.89 98.89 98.16 98.89 98.89 98.89 98.89 98.8998.89 95.20 ZIP18 99.26 99.26 98.52 99.26 99.26 99.26 99.26 99.26 99.2695.57 ZIP19 98.89 98.89 98.16 98.89 98.89 98.89 98.89 98.89 98.89 95.20ZIP20 99.26 99.26 98.52 99.26 99.26 99.26 99.26 99.26 99.26 95.57 ZIP2199.26 99.26 98.52 99.26 99.26 99.26 99.26 99.26 99.26 95.57 ZIP22 99.2699.26 98.52 99.26 99.26 99.26 99.26 99.26 99.26 95.57 ZIP23 99.26 99.2698.52 99.26 99.26 99.26 99.26 99.26 99.26 95.57 ZIP24 99.26 99.26 98.5299.26 99.26 99.26 99.26 99.26 99.26 95.57 ZIP25 99.26 99.26 98.52 99.2699.26 99.26 99.26 99.26 99.26 95.57 ZIP26 99.26 99.26 98.52 99.26 99.2699.26 99.26 99.26 99.26 95.57 ZIP27 99.26 99.26 98.52 99.26 99.26 99.2699.26 99.26 99.26 95.57 ZIP28 99.26 98.52 99.26 99.26 99.26 99.26 99.2599.26 95.57 ZIP29 99.26 98.52 99.26 99.26 99.26 99.26 99.26 99.26 95.57ZIP30 98.52 98.52 98.52 98.52 98.52 98.52 98.52 98.52 95.57 ZIP31 99.2699.26 98.52 99.26 99.26 99.26 99.26 99.26 95.57 ZIP32 99.26 99.26 98.5299.26 99.26 99.26 99.26 99.26 95.57 ZIP33 99.26 99.26 98.52 99.26 99.2699.26 99.26 99.26 95.57 ZIP34 99.26 99.26 98.52 99.26 99.26 99.26 99.2699.26 95.57 ZIP35 99.26 99.26 98.52 99.26 99.26 99.26 99.26 99.26 95.57ZIP38 99.26 99.26 98.52 99.26 99.26 99.26 99.26 99.26 95.57 ZIP39 95.5795.57 95.57 95.57 95.57 95.57 95.57 95.57 95.57

TABLE 5 Sequence Identity Matrix of 32 Novel Insecticidal Proteins FromTable 3 Identified via the IPDP and Monalysin Monalysin ZIP1 ZIP2 ZIP3ZIP5 ZIP6 ZIP8 ZIP9 ZIP10 ZIP11 ZIP12 ZIP13 Monalysin 74.17 71.59 74.1773.80 74.17 73.80 73.80 71.60 73.43 74.17 74.17 ZIP1 74.17 97.42 95.9495.20 97.05 99.63 99.26 95.96 99.26 98.89 99.26 ZIP2 71.59 97.42 93.3692.62 94.47 97.05 96.68 98.57 96.68 96.31 96.68 ZIP3 74.17 95.94 93.3694.10 95.20 95.57 95.20 91.90 95.20 94.83 95.20 ZIP5 73.80 95.20 92.6294.10 94.10 94.83 94.47 93.38 95.94 94.10 94.47 ZIP6 74.17 97.05 94.4795.20 94.10 96.68 96.31 93.01 96.31 96.68 97.05 ZIP8 73.80 99.63 97.0595.57 94.83 96.68 98.89 95.59 98.89 95.82 98.89 ZIP9 73.80 99.26 96.6895.20 94.47 96.31 98.89 95.22 98.52 98.16 98.52 ZIP10 71.60 95.96 98.5791.90 93.38 93.01 95.59 95.22 95.22 94.85 95.22 ZIP11 73.43 99.26 99.6895.20 95.94 96.31 98.89 98.52 95.22 98.16 98.52 ZIP12 74.17 98.89 96.3194.83 94.10 96.68 98.52 96.16 94.85 98.16 98.89 ZIP13 74.17 99.26 96.6895.20 94.47 97.05 98.89 98.52 95.22 98.52 98.89 ZIP17 73.80 99.26 96.5895.20 94.47 96.31 98.89 98.52 95.22 98.52 98.16 98.52 ZIP18 74.17 99.6397.05 95.94 94.83 96.68 99.26 98.89 95.59 98.89 98.52 98.89 ZIP19 73.8099.26 96.68 95.20 94.47 96.31 98.89 98.52 95.22 98.52 98.16 98.52 ZIP2073.80 99.63 97.05 95.57 94.83 96.68 99.26 98.89 95.59 98.89 98.52 98.89ZIP21 73.80 99.63 97.05 95.94 94.83 96.68 99.26 98.89 95.59 98.89 98.5298.89 ZIP22 74.54 99.63 97.05 95.57 94.83 97.42 99.26 98.89 95.59 98.8999.26 99.63 ZIP23 73.80 99.63 97.05 95.57 94.83 96.68 99.26 98.89 95.5998.89 98.52 98.89 ZIP24 73.80 99.63 97.05 95.57 94.83 96.68 99.26 98.8995.59 98.59 98.52 98.89 ZIP25 73.80 99.63 97.05 95.57 94.83 96.68 99.2698.89 95.59 98.89 98.52 98.89 ZIP26 74.17 99.63 97.05 95.57 94.83 97.0599.26 98.89 95.59 98.89 98.89 99.26 ZIP27 73.80 99.63 97.05 95.57 94.8396.68 99.26 98.89 95.59 98.89 98.52 98.89 ZIP28 74.17 99.63 97.05 95.5794.83 96.68 99.26 98.89 95.59 98.89 98.52 98.89 ZIP29 74.17 99.63 97.0595.57 94.83 96.68 99.26 98.89 95.59 98.89 98.52 98.89 ZIP30 73.80 98.8996.31 95.57 94.83 96.31 98.52 98.16 94.85 98.16 97.79 98.16 ZIP31 74.1799.63 97.05 95.57 94.83 96.68 99.26 98.89 95.59 98.89 98.52 98.89 ZIP3273.80 99.63 97.05 95.57 94.83 96.68 99.26 98.89 95.59 98.89 98.52 98.89ZIP33 74.17 99.63 97.05 95.57 94.83 96.68 99.26 99.26 95.59 98.89 98.5298.89 ZIP34 73.80 99.63 97.05 95.57 94.83 96.68 99.26 96.89 95.59 98.8998.52 96.89 ZIP35 73.80 99.63 97.05 95.57 94.83 96.68 99.26 98.89 95.5998.89 98.52 98.89 ZIP38 74.17 99.63 97.05 95.57 94.83 96.68 99.26 98.8995.59 98.89 98.52 98.89 ZIP39 74.54 95.94 93.36 94.47 99.26 94.83 95.5795.20 93.38 96.68 94.83 95.20 Monalysin ZIP17 ZIP18 ZIP19 ZIP20 ZIP21ZIP22 ZIP23 ZIP24 ZIP25 ZIP26 ZIP27 Monalysin 73.80 74.17 73.80 73.8073.80 74.54 73.80 73.80 73.80 74.17 73.80 ZIP1 74.17 99.26 99.63 99.2699.63 99.63 99.63 99.63 99.63 99.63 99.63 99.63 ZIP2 71.59 96.68 97.0596.68 97.05 97.05 97.05 97.05 97.05 97.05 97.05 97.05 ZIP3 74.17 95.2095.94 95.20 95.57 95.94 95.57 95.57 95.57 95.57 95.57 95.57 ZIP5 73.8094.47 94.83 94.47 94.83 94.83 94.83 94.83 94.83 94.83 94.83 94.83 ZIP674.17 96.31 96.68 96.31 96.65 96.68 97.42 96.68 96.65 96.68 97.05 96.68ZIP8 73.80 98.89 99.26 98.89 99.26 99.26 99.26 99.26 99.26 99.26 99.2699.26 ZIP9 73.80 98.52 98.89 98.52 98.89 98.89 98.89 98.89 98.89 98.8998.89 98.89 ZIP10 71.60 95.22 95.59 95.22 95.59 95.59 95.59 95.59 95.5995.59 95.59 95.59 ZIP11 73.43 98.52 98.89 98.52 98.89 98.89 98.89 98.8998.89 98.89 98.89 98.89 ZIP12 74.17 98.16 98.52 98.16 98.52 98.52 99.2698.52 98.52 98.52 98.89 98.52 ZIP13 74.17 98.52 98.89 98.52 98.89 98.8999.63 98.89 98.89 98.89 99.26 98.89 ZIP17 73.80 98.89 98.52 98.89 95.8998.89 98.89 98.89 98.89 98.89 98.89 ZIP18 74.17 98.89 98.89 99.26 99.2699.26 99.26 99.26 99.26 99.26 99.26 ZIP19 73.80 98.52 98.89 98.89 98.8998.89 98.89 98.89 98.89 98.89 98.89 ZIP20 73.80 98.89 99.26 98.89 99.2699.26 99.26 99.26 99.26 99.26 99.26 ZIP21 73.80 98.89 99.26 98.89 99.2699.26 99.26 99.26 99.26 99.26 99.26 ZIP22 74.54 98.89 99.26 98.89 99.2699.26 99.26 99.26 99.26 99.63 99.26 ZIP23 73.80 98.89 99.26 98.89 99.2699.26 99.26 99.26 99.26 99.26 99.26 ZIP24 73.80 98.89 99.26 98.89 99.2699.26 99.26 99.26 99.26 99.26 99.26 ZIP25 73.80 98.89 99.26 98.89 99.2699.26 99.26 99.26 99.26 99.26 99.26 ZIP26 74.17 98.89 99.26 98.89 99.2699.26 99.63 99.26 99.26 99.26 99.26 ZIP27 73.80 98.89 99.26 98.89 99.2699.26 99.26 99.26 99.26 99.26 99.26 ZIP28 74.17 98.89 99.26 98.89 99.2699.26 99.26 99.26 99.26 99.26 99.26 99.26 ZIP29 74.17 98.89 99.26 98.8999.26 99.26 99.26 99.26 99.26 99.26 99.26 99.26 ZIP30 73.80 98.16 98.5298.16 98.52 98.52 98.52 98.52 98.52 98.52 98.52 98.52 ZIP31 74.17 98.8999.26 98.89 99.26 99.26 99.26 99.26 99.26 99.26 99.26 99.26 ZIP32 73.8098.89 99.26 98.89 99.26 99.26 99.26 99.26 99.26 99.26 99.26 99.26 ZIP3374.17 98.89 99.26 98.89 99.26 99.26 99.26 99.26 99.26 99.26 99.26 99.26ZIP34 73.80 98.89 99.26 98.89 99.26 99.26 99.26 99.26 99.26 99.26 99.2699.26 ZIP35 73.80 98.89 99.26 98.89 99.26 99.26 99.26 99.26 99.26 99.2699.26 99.26 ZIP38 74.17 98.89 99.26 98.89 99.26 99.26 99.26 99.26 99.2699.26 99.26 99.26 ZIP39 74.54 95.20 95.57 95.20 95.57 95.57 95.57 95.5795.57 95.57 95.57 95.57 Monalysin ZIP28 ZIP29 ZIP30 ZIP31 ZIP32 ZIP33ZIP34 ZIP35 ZIP38 ZIP39 Monalysin 74.17 74.17 73.80 74.17 73.80 74.1773.80 73.80 74.17 74.54 ZIP1 74.17 99.63 99.63 98.89 99.63 99.63 99.6399.63 99.63 99.63 95.94 ZIP2 71.59 97.05 97.05 96.31 97.05 97.05 97.0597.05 97.05 97.05 93.36 ZIP3 74.17 95.57 95.57 95.57 95.57 95.57 95.5795.57 95.57 95.57 94.47 ZIP5 73.80 94.83 94.83 94.83 94.83 94.83 94.8394.83 94.83 94.83 99.26 ZIP6 74.17 96.68 96.68 96.31 96.68 96.68 96.6896.68 96.68 96.68 94.83 ZIP8 73.80 99.26 99.26 98.52 99.26 99.26 99.2699.26 99.26 99.26 95.57 ZIP9 73.80 98.89 98.89 98.16 98.89 98.89 99.2698.89 98.89 98.89 95.20 ZIP10 71.60 95.59 95.59 94.85 95.59 95.59 95.5995.59 95.59 95.59 93.38 ZIP11 73.43 98.89 98.89 98.16 98.89 98.89 98.8998.89 98.89 98.89 96.68 ZIP12 74.17 98.52 98.52 97.79 98.52 98.52 98.5298.52 98.52 98.52 94.83 ZIP13 74.17 98.89 98.89 98.16 98.89 98.89 98.8998.89 98.89 98.89 95.20 ZIP17 73.80 98.89 98.89 98.16 98.89 98.89 98.8998.89 98.89 98.89 95.20 ZIP18 74.17 99.26 99.26 98.52 99.26 99.26 99.2699.26 99.26 99.26 95.57 ZIP19 73.80 98.89 98.89 98.16 98.89 98.89 98.8998.89 98.89 98.89 95.20 ZIP20 73.80 99.26 99.26 98.52 99.26 99.26 99.2699.26 99.26 99.26 95.57 ZIP21 73.80 99.26 99.26 98.52 99.26 99.26 99.2699.26 99.26 99.26 95.57 ZIP22 74.54 99.26 99.26 98.52 99.26 99.26 99.2699.26 99.26 99.26 95.57 ZIP23 73.80 99.26 99.26 98.52 99.26 99.26 99.2699.26 99.26 99.26 95.57 ZIP24 73.80 99.26 99.26 98.52 99.26 99.26 99.2699.26 99.26 99.26 95.57 ZIP25 73.80 99.26 99.26 98.52 99.26 99.26 99.2699.26 99.26 99.26 95.57 ZIP26 74.17 99.26 99.26 98.52 99.26 99.26 99.2699.26 99.26 99.26 95.57 ZIP27 73.80 99.26 99.26 98.52 99.26 99.26 99.2699.26 99.26 99.26 95.57 ZIP28 74.17 99.26 98.52 99.26 99.26 99.26 99.2699.26 99.26 95.57 ZIP29 74.17 99.26 98.52 99.26 99.26 99.26 99.26 99.2699.26 95.57 ZIP30 73.80 98.52 98.52 98.52 98.52 98.52 98.52 98.52 98.5295.57 ZIP31 74.17 99.26 99.26 98.52 99.26 99.26 99.26 99.26 99.26 95.57ZIP32 73.80 99.26 99.26 98.52 99.26 99.26 99.26 99.26 99.26 95.57 ZIP3374.17 99.26 99.26 98.52 99.26 99.26 99.26 99.26 99.26 95.57 ZIP34 73.8099.26 99.26 98.52 99.26 99.26 99.26 99.26 99.26 95.57 ZIP35 73.80 99.2699.26 98.52 99.26 99.26 99.26 99.26 99.26 95.57 ZIP38 74.17 99.26 99.2698.52 99.26 99.26 99.26 99.26 99.26 95.57 ZIP39 74.54 95.57 95.57 95.5795.57 95.57 95.57 95.57 95.57 95.57

A multiple sequence alignment for the eight novel insecticidal proteinsfrom Table 3 (i.e., ZIP1, ZIP2, ZIP6, ZIP8, ZIP9, ZIP10, ZIP11, andZIP12) can be found in FIG. 3, with a corresponding phylogenetic treefound in FIG. 5. A multiple sequence alignment of the eight novelinsecticidal proteins from Table 3 (i.e., ZIP1, ZIP2, ZIP6, ZIP8, ZIP9,ZIP10, ZIP11, and ZIP12), plus monalysin, can be found in FIG. 4, with acorresponding phylogenetic tree found in FIG. 6.

Example 3: Insecticidal Proteins—Lysate Insect Feeding Assays

Lysate from bacteria expressing either ZIP1 (SEQ ID NO: 2), or an emptyvector control, were compared to water in an insect feeding assayutilizing Brown Marmorated Stinkbugs (Halyomorpha halys).

2 cm of a wick was moistened with 0.5 mL of lysate, or water, and placedin a plate with 2 first instar nymphs (one observation). 8 observationsper lysate, or water, were tested for a total of 16 insects per sample.

Mortality was measured after 7 days.

Insects allowed to ingest bacterial lysate from cells containing emptyvector showed similar levels of mortality, as those ingesting water.

Insects allowed to ingest bacterial lysate expressing ZIP1 (SEQ ID NO:2) showed 100% mortality in this assay. These results are illustrated inFIG. 10.

Consequently, an insecticidal protein as taught herein demonstratessignificant insecticidal activity against a member of the OrderHemiptera and family Pentatomidae.

Example 4: Insecticidal Proteins—Purified Protein Insect Feeding Assays

From the 36 novel insecticidal proteins represented in Table 3, weengineered 10 protein sequences to be fused with an N-terminal 6×Hisprotein tag for purification: a) SEQ ID NO: 2 that is ZIP1, b) SEQ IDNO: 4 that is ZIP2, c) SEQ ID NO: 8 that is ZIP4, d) SEQ ID NO: 12 thatis ZIP6, e) SEQ ID NO: 14 that is ZIP8, f) SEQ ID NO: 16 that is ZIP9,g) SEQ ID NO: 18 that is ZIP10, h) SEQ ID NO: 20 that is ZIP11, i) SEQID NO: 22 that is ZIP12 and i) SEQ ID NO: 26 that is ZIP16. Lysate frombacteria expressing these 10 tagged proteins were incubated with Ni-NTAbeads (Qiagen) to specifically bind the proteins. These proteins wereeluted from the beads, dialyzed and used for insect feeding assayutilizing Brown Marmorated Stinkbugs (Halyomorpha halys).

For Brown Marmorated Stinkbugs, 2 cm of a wick was moistened with 0.5 mLof purified protein, or buffer control or water, and placed in a platewith 2 first instar nymphs (one observation). 8 observations per lysate,or water, were tested for a total of 16 insects per sample.

Mortality was measured after 5 days.

Insects allowed to ingest these 10 purified proteins showed varyingdegrees of mortality in this assay. These results are illustrated inFIG. 7.

Furthermore, of the ten tested proteins, three purified proteins (ZIP1,ZIP2, and ZIP4) of varying dilutions were used for replicate insectfeeding assays with Brown Marmorated Stinkbugs to establish a LC50 valuethrough Probit Analysis. These results are illustrated in FIG. 8.

Consequently, these insecticidal proteins as taught herein demonstratessignificant insecticidal activity against a member of the OrderHemiptera and family Pentatomidae.

To determine the effective range of insects for these insecticidalproteins, we performed insect bioassays against members of the two othermajor Orders of insects—Fall Armyworm (Spodoptera frugiperda) from theorder Lepidoptera and Southern Corn Rootworm (Diabroticaundecimpunctata) from the order Coleoptera. Briefly, for Fall armyworm,warm multispecies insect diet is dispensed into standard 128-wellbioassay trays at a rate of 1.0 ml/well which provides a surface area of1.5 cm² within each well. Each test well was treated by applying 40 μlof purified protein onto the diet surface. Once the application hasdried, one neonate fall armyworm larva is placed into each well. Theassay consists of 16 individual wells per treatment. Buffer-only treatedwells serve as the negative control. Growth by weight was measured after7 days.

The methods used for Southern Corn Rootworm were similar to those usedfor Fall Armyworm except a Southern Corn Rootworm specific diet wasused.

Insects allowed to ingest purified protein expressing ZIP1, ZIP2 andZIP4 showed varying degrees of growth inhibition in this assay. Theresults for Fall Armyworm are illustrated in FIG. 9A and the results forSouthern Corn Rootworm are illustrated in FIG. 9B.

Consequently, these insecticidal proteins as taught herein demonstratessignificant growth inhibitory activity against a members of the OrderLepidoptera and family Noctuidae, and the Order Coleoptera and familyChrysomelidae.

Example 5: Identification of Novel Insecticidal Proteins UtilizingHomology and Profile/HMM Methods (IPDP—HMM Construction)

As discussed previously, the IPDP can optionally involve the utilizationof a HMM algorithm and modeling procedure. This procedure allows for thedevelopment of a HMM profile from the discovered insecticidal proteinsequences that identifies genes encoding monalysin-like insecticidalproteins that would not be possible to identify using some methods ofthe art, e.g. BLAST.

An example of the HMM process is described below and was built usingeight insecticidal proteins discovered via the IPDP and found in Table 3and highlighted in FIG. 4. An example HMM built using eight insecticidalproteins identified using methods described herein is provided in Table6. These proteins have the amino acid sequences shown in: a) SEQ ID NO:2 that is ZIP1, b) SEQ ID NO: 4 that is ZIP2, c) SEQ ID NO: 12 that isZIP6, d) SEQ ID NO: 14 that is ZIP8, e) SEQ ID NO: 16 that is ZIP9, f)SEQ ID NO: 18 that is ZIP10, g) SEQ ID NO: 20 that is ZIP11 and h) SEQID NO: 22 that is ZIP12.

The model was constructed using the HMMER software (Version 3.1b2;February 2015) and the output model can be found in Table 6. The HMMutilized the aforementioned eight sequences to create a model of what a“monalysin-like” sequence (based on the eight utilized sequences) wouldentail. Now, based on the HMM, it is possible to analyze future putativeinsecticidal proteins discovered with the IPDP to determine thelikelihood that the newly discovered sequences are a “monalysin-like”sequence.

Example 6: Transformed Plants

Experiments were conducted to transform a plant of interest (e.g., maizeand soybean) for stable expression of insecticidal proteins disclosedherein. Three ZIP proteins, ZIP1, ZIP2 and ZIP4, demonstrated to killinsects in in vitro bioassays (see, e.g., Example 4) were selected to betransformed into two crop plants of interest—soybean and maize. Thesesequences were codon optimized for optimal expression in the plants ofinterest and synthesized and cloned into specific expression vectors forboth soybean and maize.

Soybean

Soybean seeds are surface sanitized in 20% Clorox, rinsed with sterilewater, then primed by allowing them to sit for 2 hours at roomtemperature. Seeds are then imbibed in Germination Medium overnight.Meristem explants are prepared the next day by removing seed coats andcotyledons from the seed. Meristem explants are then either dried undera variety of conditions, or used fresh. For biolistic DNA delivery,vectors are coated onto gold particles (0.6 μm) for particle bombardmentvia the Bio-Rad PDS-1000 Helium gun according to standard protocol. Forparticle bombardment, explants are pre-cultured overnight, bombarded,allowed to rest, then transferred to selection. Shoots fromspectinomycin resistant plantlets are harvested and rooted on rootingmedia containing IAA and spectinomycin. Rooted plants are transplantedto soil and grown in the greenhouse to produce T1 seed.

Maize

Immature embryos (1.5-2.0 mm) from greenhouse or field grown Hi-II maizeare dissected out in a sterile hood. Embryos are co-cultured for 1-2days at 23° C. in the dark with Agrobacterium strain AGL-1 at a finalOD660 of ˜0.4 axis side down on solid co-cultivation medium. Embryos arethen transferred to solid induction media, axis side down, and incubatedat 28° C. for 5 days in the dark. Embryos are then transferred to solidselection 1 medium (bialaphos) and incubated at 28° C. for two weeks inthe dark. Embryos are transferred to solid selection medium 2(bialaphos) and incubated at 28° C. in the dark. Resistant callusforming embryos are transferred every two weeks until diameter is about1.5-2 cm. Transfer resistant calli to solid regeneration medium 1 at 28°C. for 2 weeks. Transfer calli to regeneration medium 2 at 28° C. under16 hour photoperiod until shoots and roots develop. Transfer plantletsto soil and grow to maturity.

To select plants expressing insecticidal proteins taught in thisdisclosure, antibiotic selection media was used for further growth ofregenerated shoots from the calli, and the regenerated plants weretested for the expression of insecticidal proteins disclosed herein. Asshown in FIG. 11, expression of ZIP1, ZIP2 and ZIP4 proteins wasdetected by Western Blotting with a ZIP-specific antibody from leaves ofthe transformed soybean plants (Lanes 2-10 and 12-13 of FIG. 11). Also,ZIP2 protein expression was confirmed in leaves of maize plantstransformed with expression vector specific for ZIP2 (Lanes 15 and 16 ofFIG. 11). Other transgenic soybean and maize plants expressinginsecticidal proteins found in Table 3 are being tested for expressionof the proteins of interest.

Furthermore, insect feeding assays are being carried out with a part(e.g. leaves, stems, roots, flowers, fruits, seeds, or seedlings) of thetransformed plants stably expressing the insecticidal proteins ofinterest, including ZIP1, ZIP2, and ZIP4 shown in Example 6 as well asother ZIP proteins found in Table 3.

TABLE 6 HMMER3/f [3.1b2] | February 2015 NAME zips LENG 272 ALPH aminoRF no MM | no CONS yes CS no MAP yes DATE Thu Feb 21 21:13:29 2019 NSEQ8 EFFN 0.417969 CKSUM 1653877865 STATS LOCAL MSV −11.4830 0.70210 STATSLOCAL VITERBI −11.8487 0.70210 STATS LOCAL FORWARD  −5.1315 0.70210 A CD E F G H HMM m -> m m -> i m -> d i -> m i -> i d -> m d -> d I K L M NP Q R S T V W Y COMPO 2.49688 4.38085 2.89420 2.83896 3.17739 2.746933.76314 2.84203 2.81709 2.55776 3.86213 3.04964 3.45042 3.03015 3.106702.49841 2.71919 2.59721 4.46810 3.23303 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.808394.53074 0.61958 0.77255 0.00000 *  1 2.92069 4.48020 3.98129 3.571133.18275 3.78627 4.32118 2.35208 3.34223 1.78828 1.50662 3.85616 4.247613.72903 3.55483 3.26253 3.22531 2.35043 5.01429 3.76968 1 m - - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 2 2.38088 4.22532 3.37373 3.12452 3.91469 3.06626 4.14640 3.034113.06620 2.89981 3.95571 3.34319 3.76550 3.46463 3.32709 2.58160 1.285462.73596 5.35824 4.13176 2 t - - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.808394.53074 0.61958 0.77255 0.48576 0.95510  3 2.98131 4.41980 4.241623.85620 3.33257 3.94480 4.59720 1.16111 3.68296 1.93351 3.27971 4.110594.43351 4.03877 3.88695 3.48513 3.28221 1.84862 5.20905 3.94078 3i - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.485760.95510  4 2.91557 4.87384 3.08714 2.72252 4.24557 3.41039 3.727463.67115 1.12970 3.25103 4.21733 3.14931 3.91643 2.92363 2.42483 2.962603.17436 3.38305 5.32773 4.13178 4 k - - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.033513.80839 4.53074 0.61958 0.77255 0.48576 0.95510  5 2.89992 4.993762.46525 1.12027 4.33240 3.23467 3.81790 3.77361 2.69052 3.40180 4.380242.90101 3.83668 3.03593 3.09149 2.88987 3.20420 3.46469 5.51622 4.240175 e - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.772550.48576 0.95510  6 2.89992 4.99376 2.46525 1.12027 4.33240 3.234673.81790 3.77361 2.69052 3.40180 4.38024 2.90101 3.83668 3.03593 3.091492.88987 3.20420 3.46469 5.51622 4.24017 6 e - - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510  7 3.084694.56681 4.08583 3.72691 3.11381 3.89935 4.40279 2.31622 3.49522 0.958453.15956 4.00841 4.34677 3.87896 3.68117 3.46490 3.38246 2.33662 4.968333.67637 7 l - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.619580.77255 0.48576 0.95510  8 2.19939 4.16075 3.07524 2.86856 4.198422.26559 4.04696 3.66282 3.00754 3.34450 4.18714 3.11776 3.61262 3.312633.33572 1.44764 2.67538 3.13195 5.52641 4.27542 8 s - - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510  92.73569 4.88388 2.64985 2.40631 4.13527 3.28203 3.66153 3.71230 2.335993.27126 4.16322 2.63239 3.81944 1.71852 2.66538 2.74932 3.01644 3.372165.36856 4.00601 9 q - - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.530740.61958 0.77255 0.48576 0.95510  10 2.67118 4.43758 3.27171 3.102684.10073 3.12878 4.18431 3.63096 3.15473 3.26429 4.31243 3.41512 0.955883.54553 3.40634 2.83701 3.10805 3.28352 5.30361 4.22102 10 p - - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 11 2.83288 4.81384 2.86375 2.61915 3.97341 3.32530 3.77632 3.595812.44483 3.10440 4.13570 3.07769 3.87889 1.35270 2.72959 2.88411 3.130683.32998 5.25766 3.94763 11 q - - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.808394.53074 0.61958 0.77255 0.48576 0.95510  12 2.28599 4.20474 3.153132.96740 4.01316 2.93592 4.07832 3.52777 3.03676 3.25353 4.18606 3.201533.66974 3.39147 3.32378 1.19199 2.76159 3.07699 5.39114 4.09669 12s - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.485760.95510  13 2.92923 4.75570 3.06737 2.82225 3.24301 3.39609 1.285223.62204 2.66722 3.12353 4.16260 3.23362 3.94584 3.18806 2.93608 3.000913.23101 3.35853 4.70870 3.19598 13 h - - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.033513.80839 4.53074 0.61958 0.77255 0.48576 0.95510  14 2.28599 4.204743.15313 2.96740 4.01316 2.93592 4.07832 3.52777 3.03676 3.25353 4.186063.20153 3.66974 3.39147 3.32378 1.19199 2.76159 3.07699 5.39114 4.0966914 s - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.772550.48576 0.95510  15 2.75784 4.31972 4.03530 3.69790 3.50486 3.655974.51118 2.05876 3.56463 2.20413 3.48128 3.89758 4.22110 3.92973 3.783023.15664 3.12163 1.11004 5.27795 4.00490 15 v - - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510  16 2.899924.99376 2.46525 1.12027 4.33240 3.23467 3.81790 3.77361 2.69052 3.401804.38024 2.90101 3.83668 3.03593 3.09149 2.88987 3.20420 3.46469 5.516224.24017 16 e - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.619580.77255 0.48576 0.95510  17 3.08469 4.56681 4.08583 3.72691 3.113813.89935 4.40279 2.31622 3.49522 0.95845 3.15956 4.00841 4.34677 3.878963.68117 3.46490 3.38246 2.33662 4.96833 3.67637 17 l - - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510  182.92310 5.03735 1.01454 2.30009 4.40640 3.16420 3.87837 3.98050 2.950993.60099 4.57912 2.85261 3.81266 3.11848 3.45728 2.89377 3.26085 3.627045.58736 4.29177 18 d - - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.530740.61958 0.77255 0.48576 0.95510  19 2.83288 4.81384 2.86375 2.619153.97341 3.32530 3.77632 3.59581 2.44483 3.10440 4.13570 3.07769 3.878891.35270 2.72959 2.88411 3.13068 3.32998 5.25766 3.94763 19 q - - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 20 3.08469 4.56681 4.08583 3.72691 3.11381 3.89935 4.40279 2.316223.49522 0.95845 3.15956 4.00841 4.34677 3.87896 3.68117 3.46490 3.382462.33662 4.96833 3.67637 20 l - - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.808394.53074 0.61958 0.77255 0.48576 0.95510  21 2.83288 4.81384 2.863752.61915 3.97341 3.32530 3.77632 3.59581 2.44483 3.10440 4.13570 3.077693.87889 1.35270 2.72959 2.88411 3.13068 3.32998 5.25766 3.94763 21q - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.485760.95510  22 2.75784 4.31972 4.03530 3.69790 3.50486 3.65597 4.511182.05876 3.56463 2.20413 3.48128 3.89758 4.22110 3.92973 3.78302 3.156643.12163 1.11004 5.27795 4.00490 22 v - - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.033513.80839 4.53074 0.61958 0.77255 0.48576 0.95510  23 2.58929 4.369403.24976 3.14188 4.28221 0.83523 4.26473 3.86748 3.31425 3.52941 4.505783.41443 3.75846 3.64375 3.55601 2.75963 3.06560 3.40832 5.39294 4.3824523 g - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.772550.48576 0.95510  24 2.89992 4.99376 2.46525 1.12027 4.33240 3.234673.81790 3.77361 2.69052 3.40180 4.38024 2.90101 3.83668 3.03593 3.091492.88987 3.20420 3.46469 5.51622 4.24017 24 e - - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510  25 2.757844.31972 4.03530 3.69790 3.50486 3.65597 4.51118 2.05876 3.56463 2.204133.48128 3.89758 4.22110 3.92973 3.78302 3.15664 3.12163 1.11004 5.277954.00490 25 v - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.619580.77255 0.48576 0.95510  26 2.28599 4.20474 3.15313 2.96740 4.013162.93592 4.07832 3.52777 3.03676 3.25353 4.18606 3.20153 3.66974 3.391473.32378 1.19199 2.76159 3.07699 5.39114 4.09669 26 s - - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.24354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510  271.11031 4.16266 3.39952 3.18951 3.99036 2.98856 4.21886 3.10649 3.204142.99084 4.03105 3.35470 3.72187 3.55158 3.45819 2.51417 2.78716 2.772115.42402 4.21744 27 a - - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.530740.61958 0.77255 0.48576 0.95510  28 2.93567 4.77649 3.42005 2.946504.11486 3.43409 3.78227 3.65333 2.21804 3.18106 4.18431 3.31043 3.941053.60701 1.08112 3.02376 3.20684 3.38040 5.22475 4.05755 28 r - - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 29 2.89992 4.99376 2.46525 1.12027 4.33240 3.23467 3.81790 3.773612.69052 3.40180 4.38024 2.90101 3.83668 3.03593 3.09149 2.88987 3.204203.46469 5.51622 4.24017 29 e - - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.808394.53074 0.61958 0.77255 0.48576 0.95510  30 1.11031 4.16266 3.399523.18951 3.99036 2.98856 4.21886 3.10649 3.20414 2.99084 4.03105 3.354703.72187 3.55158 3.45819 2.51417 2.78716 2.77211 5.42402 4.21744 30a - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.485760.95510  31 3.08469 4.56681 4.08583 3.72691 3.11381 3.89935 4.402792.31622 3.49522 0.95845 3.15956 4.00841 4.34677 3.87896 3.68117 3.464903.38246 2.33662 4.96833 3.67637 31 l - - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.033513.80839 4.53074 0.61958 0.77255 0.48576 0.95510  32 2.38088 4.225323.37373 3.12452 3.91469 3.06626 4.14640 3.03411 3.06620 2.89981 3.955713.34319 3.76550 3.46463 3.32709 2.58160 1.28546 2.73596 5.35824 4.1317632 t - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.772550.48576 0.95510  33 2.00874 4.12949 3.18245 2.95794 4.11369 2.886904.07663 3.47283 3.02570 3.23256 4.11484 3.16881 3.62621 3.35625 3.329011.36684 2.66847 3.00171 5.49017 4.22951 33 s - - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510  34 2.672664.66469 2.67738 2.55713 4.01837 3.14279 3.87335 3.71632 2.77246 3.370974.32772 1.27686 3.79220 3.14721 3.12388 2.74309 3.05315 3.34503 5.331893.97225 34 n - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.619580.77255 0.48576 0.95510  35 3.17889 4.60008 4.15032 3.85387 1.122823.88119 3.85245 2.71188 3.76280 2.09750 3.45251 3.94779 4.34224 3.948873.89833 3.45132 3.48052 2.69761 4.12292 2.49894 35 f - - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510  361.11031 4.16266 3.39952 3.18951 3.99036 2.98856 4.21886 3.10649 3.204142.94084 4.03105 3.35470 3.72187 3.55158 3.45819 2.51417 2.78716 2.772115.42402 4.21744 36 a - - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.530740.61958 0.77255 0.48576 0.95510  37 2.58929 4.36940 3.24976 3.141884.28221 0.83523 4.26473 3.86748 3.31425 3.52941 4.50578 3.41443 3.758463.64375 3.55601 2.75963 3.06560 3.40832 5.39294 4.38245 37 g - - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 38 2.28599 4.20474 3.15313 2.96740 4.01316 2.93592 4.07832 3.527773.03676 3.25353 4.18606 3.20153 3.66974 3.39147 3.32378 1.19199 2.761593.07699 5.39114 4.09669 38 s - - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.808394.53074 0.61958 0.77255 0.48576 0.95510  39 3.17889 4.60008 4.150323.85387 1.12282 3.88119 3.85245 2.71188 3.76280 2.09750 3.45251 3.947794.34224 3.94887 3.89833 3.45132 3.48052 2.69761 4.12292 2.49894 39f - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.485760.95510  40 2.92310 5.03735 1.01454 2.30009 4.40640 3.16420 3.878373.98050 2.95099 3.60099 4.57912 2.85261 3.81266 3.11848 3.45728 2.893773.26085 3.62704 5.58736 4.29177 40 d - - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.033513.80839 4.53074 0.61958 0.77255 0.48576 0.95510  41 2.83288 4.813842.86375 2.61915 3.97341 3.32530 3.77632 3.59581 2.44483 3.10440 4.135703.07769 3.87889 1.35270 2.72959 2.88411 3.13068 3.32998 5.25766 3.9476341 q - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.772550.48576 0.95510  42 3.17889 4.60008 4.15032 3.85387 1.12282 3.881193.85245 2.71188 3.76280 2.09750 3.45251 3.94779 4.34224 3.94887 3.898333.45132 3.48052 2.69761 4.12292 2.49894 42 f - - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510  43 2.671184.43758 3.27171 3.10268 4.10073 3.12878 4.18431 3.63096 3.15473 3.264294.31243 3.41512 0.95588 3.54553 3.40634 2.83701 3.10805 3.28352 5.303614.22102 43 p - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.619580.77255 0.48576 0.95510  44 2.38088 4.22532 3.37373 3.12452 3.914693.06626 4.14640 3.03411 3.06620 2.89981 3.95571 3.34319 3.76550 3.464633.32709 2.58160 1.28546 2.73596 5.35824 4.13176 44 t - - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510  452.91557 4.87384 3.08714 2.72252 4.24557 3.41039 3.72746 3.67115 1.129703.25103 4.21733 3.14931 3.91643 2.92363 2.42483 2.96260 3.17436 3.383055.32773 4.13178 45k - - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.40347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.530740.61958 0.77255 0.48576 0.95510  46 2.28599 4.20474 3.15313 2.967404.01316 2.93592 4.07832 3.52777 3.03676 3.25353 4.18606 3.20153 3.669743.39147 3.32378 1.19199 2.76159 3.07699 5.39114 4.09669 46 s - - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 47 2.58929 4.36940 3.24976 3.14188 4.28221 0.83523 4.26473 3.867483.31425 3.52941 4.50578 3.41443 3.75846 3.64375 3.55601 2.75963 3.065603.40832 5.39294 4.38245 47 g - - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.63351 3.808394.53074 0.61958 0.77255 0.48576 0.95510  48 2.20984 4.16363 3.087192.88717 4.17468 2.53250 4.05422 3.64534 3.01073 3.33804 4.19084 3.131033.62005 3.32767 3.32855 1.33690 2.68690 3.12426 5.50843 4.25298 48s - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.485760.95510  49 3.17889 4.60008 4.15032 3.85387 1.12282 3.88119 3.852452.71188 3.76280 2.09750 3.45251 3.94779 4.34224 3.94887 3.89833 3.451323.48052 2.69761 4.12292 2.49894 49 f - - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.033513.80839 4.53074 0.61958 0.77255 0.48576 0.95510  50 2.89992 4.993762.46525 1.12027 4.33240 3.23467 3.81790 3.77361 2.69052 3.40180 4.380242.90101 3.83668 3.03593 3.09149 2.88987 3.20420 3.46469 5.51622 4.2401750 e - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.772550.48576 0.95510  51 2.98131 4.41980 4.24162 3.85620 3.33257 3.994804.59720 1.16111 3.68296 1.93351 3.27971 4.11059 4.43351 4.03877 3.886953.48513 3.28221 1.84862 5.20905 3.94078 51 i - - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510  52 2.923105.03735 1.01454 2.30009 4.40640 3.16420 3.87837 3.98050 2.95099 3.600994.57912 2.85261 3.81266 3.11848 3.45728 2.89377 3.26085 3.62704 5.587364.29177 52 d - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.619580.77255 0.48576 0.95510  53 2.91557 4.87384 3.08714 2.72252 4.245573.41039 3.72746 3.67115 1.12970 3.25103 4.21733 3.14931 3.91643 2.923632.42483 2.96260 3.17436 3.38305 5.32773 4.13178 53 k - - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510  543.15726 4.67219 3.74475 3.46341 2.29976 3.75634 3.59289 3.17566 3.322782.64581 3.85778 3.64645 4.23235 3.64422 3.51908 3.29987 3.44402 3.043383.94061 1.07444 54 y - - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24640 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.530740.61958 0.77255 0.48576 0.95510  55 3.08469 4.56681 4.08583 3.726913.11381 3.89935 4.40279 2.31622 3.49522 0.95845 3.15956 4.00841 4.346773.87896 3.68117 3.46490 3.38246 2.33662 4.96833 3.67637 55 l - - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 56 3.08469 4.56681 4.08583 3.72691 3.11381 3.89935 4.40279 2.316223.49522 0.95845 3.15956 4.00841 4.34677 3.87896 3.68117 3.46490 3.382462.33662 4.96833 3.67637 56 l - - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.808394.53074 0.61958 0.77255 0.48576 0.95510  57 2.67266 4.66469 2.677382.55713 4.01837 3.14279 3.87335 3.71632 2.77246 3.37097 4.32772 1.276863.79220 3.14721 3.12388 2.74309 3.05315 3.34503 5.33189 3.97225 57n - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.485760.95510  58 3.15726 4.67219 3.74475 3.46341 2.29976 3.75634 3.592893.17566 3.32278 2.64581 3.85778 3.64645 4.23235 3.64422 3.51908 3.299873.44402 3.04338 3.94061 1.07944 58 y - - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.033513.80839 4.53074 0.61958 0.77255 0.48576 0.95510  59 1.11031 4.162663.39952 3.18951 3.99036 2.98856 4.21886 3.10649 3.20414 2.99084 4.031053.35470 3.72187 3.55158 3.45819 2.51417 2.78716 2.77211 5.42402 4.2174459 a - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.772550.48576 0.95510  60 2.92310 5.03735 1.01454 2.30009 4.40640 3.164203.87837 3.98050 2.95099 3.60099 4.57912 2.85261 3.81266 3.11848 3.457282.89377 3.26085 3.62704 5.58736 4.29177 60 d - - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.94347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510  61 2.671184.43758 3.27171 3.10268 4.10073 3.12878 4.18431 3.63096 3.15473 3.264294.31243 3.41512 0.95588 3.54553 3.40634 2.83701 3.10805 3.28352 5.303614.22102 61 p - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.619580.77255 0.48576 0.95510  62 2.91557 4.87384 3.08714 2.72252 4.245573.41039 3.72746 3.67115 1.12970 3.25103 4.21733 3.14931 3.91643 2.923632.42483 2.96260 3.17436 3.38305 5.32773 4.13178 62 k - - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510  632.91557 4.87384 3.08714 2.72252 4.24557 3.41039 3.72746 3.67115 1.129703.25103 4.21733 3.14931 3.91643 2.92363 2.42483 2.96260 3.17436 3.383055.32773 4.13178 63 k - - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.530740.61958 0.77255 0.48576 0.95510  64 2.58929 4.36940 3.24976 3.141884.28221 0.83523 4.26473 3.86748 3.31425 3.52941 4.50578 3.41443 3.758463.64375 3.55601 2.75963 3.06560 3.40832 5.39294 4.38245 64 g - - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 65 2.51796 1.08037 4.02002 3.74616 3.75363 3.15316 4.40750 2.888103.57311 2.80586 3.92566 3.75027 3.85502 3.92202 3.70837 2.78924 2.979542.63671 5.18182 4.04137 65 c - - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.808394.53074 0.61958 0.77255 0.48576 0.95510  66 2.86633 4.50319 3.672923.18234 2.80653 3.61109 3.68768 3.06521 2.75280 2.59879 3.66030 3.468494.05108 3.28633 2.60484 3.05056 3.12115 2.88599 1.73785 2.82731 66w - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.485760.95510  67 3.08469 4.56681 4.08583 3.72691 3.11381 3.89935 4.402792.31622 3.49522 0.95845 3.15956 4.00841 4.34677 3.87896 3.68117 3.464903.38246 2.33662 4.96833 3.67637 67 l - - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.033513.80839 4.53074 0.61958 0.77255 0.48576 0.95510  68 2.92310 5.037351.01454 2.30009 4.40640 3.16420 3.87837 3.98050 2.95099 3.60099 4.579122.85261 3.81266 3.11848 3.45728 2.89377 3.26085 3.62704 5.58736 4.2917768 d - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.772550.48576 0.95510  69 2.58929 4.36940 3.24976 3.14188 4.28221 0.835234.26473 3.86748 3.31425 3.52941 4.50578 3.41443 3.75846 3.64375 3.556012.75963 3.06560 3.40832 5.39294 4.38245 69 g - - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510  70 2.757844.31972 4.03530 3.69790 3.50486 3.65597 4.51118 2.05876 3.56463 2.204133.48128 3.89758 4.22110 3.92973 3.78302 3.15664 3.12163 1.11004 5.277954.00490 70 v - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.619580.77255 0.48576 0.95510  71 2.38088 4.22532 3.37373 3.12452 3.914693.06626 4.14640 3.03411 3.06620 2.89981 3.95571 3.34319 3.76550 3.464633.32709 2.58160 1.28546 2.73596 5.35824 4.13176 71 t - - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510  722.75784 4.31972 4.03530 3.69790 3.50486 3.65597 4.51118 2.05876 3.564632.20413 3.48128 3.89758 4.22110 3.92973 3.78302 3.15664 3.12163 1.110045.27795 4.00490 72 v - - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.530740.61958 0.77255 0.48576 0.95510  73 3.15726 4.67219 3.74475 3.463412.29976 3.75634 3.59289 3.17566 3.32278 2.64581 3.85778 3.64645 4.232353.64422 3.51908 3.29987 3.44402 3.04338 3.94061 1.07944 73 y - - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 74 2.58929 4.36940 3.24976 3.14188 4.28221 0.63523 4.26473 3.867483.31425 3.52941 4.50578 3.41443 3.75846 3.64375 3.55601 2.75963 3.065603.40832 5.39294 4.38245 74 g - - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.808394.53074 0.61958 0.77255 0.48576 0.95510  75 2.92310 5.03735 1.014542.30009 4.40640 3.16420 3.87837 3.98050 2.95099 3.60099 4.57912 2.852613.81266 3.11848 3.45728 2.89377 3.26085 3.62704 5.58736 4.29177 75d - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.485760.95510  76 2.98131 4.41980 4.24162 3.85620 3.33257 3.99480 4.597201.16111 3.68296 1.93351 3.27971 4.11059 4.43351 4.03877 3.88695 3.485133.28221 1.84862 5.20905 3.94078 76 l - - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.033513.80839 4.53074 0.61958 0.77255 0.48576 0.95510  77 3.15726 4.672193.74475 3.46341 2.29976 3.75634 3.59289 3.17566 3.32278 2.64581 3.857783.64645 4.23235 3.64422 3.51908 3.29987 3.44402 3.04338 3.94061 1.0794477 y - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.772550.48576 0.95510  78 2.98131 4.41980 4.24162 3.85620 3.33257 3.994804.59720 1.16111 3.68296 1.93351 3.27971 4.11059 4.43351 4.03877 3.886953.48513 3.28221 1.84862 5.20905 3.94078 78 i - - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510  79 2.589294.36940 3.24976 3.14188 4.28221 0.83523 4.26473 3.86748 3.31425 3.529414.50578 3.41443 3.75846 3.64375 3.55601 2.75963 3.06560 3.40832 5.392944.38245 79 g - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.619580.77255 0.48576 0.95510  80 2.91557 4.87384 3.08714 2.72252 4.245573.41039 3.72746 3.67115 1.12970 3.25103 4.21733 3.14931 3.91643 2.923632.42483 2.96260 3.17436 3.38305 5.32773 4.11178 80 k - - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510  812.83288 4.81384 2.86375 2.61915 3.97341 3.32530 3.77632 3.59581 2.444833.10440 4.13570 3.07769 3.87889 1.35270 2.72959 2.88411 3.13068 3.329985.25766 3.94763 81 q - - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.530740.61958 0.77255 0.48576 0.95510  82 2.67266 4.66469 2.67738 2.557134.61837 3.14279 3.87335 3.71632 2.77246 3.37097 4.32772 1.27686 3.792203.14721 3.12388 2.74309 3.05315 3.34503 5.33189 3.97225 82 n - - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 83 3.23357 4.63032 3.99599 3.68726 2.60690 3.66065 3.82592 3.255713.41132 2.66106 3.89235 3.84955 4.17341 3.80241 3.55653 3.43778 3.531063.14428 1.02517 2.61260 83 w - - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.808394.53074 0.61958 0.77255 0.48576 0.95510  84 2.58929 4.36940 3.249763.14188 4.28221 0.83523 4.26473 3.86748 3.31425 3.52941 4.50578 3.414433.75846 3.64375 3.55601 2.75963 3.06560 3.40832 5.39294 4.38245 84g - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.485760.95510  85 2.38088 4.22532 3.37373 3.12452 3.91469 3.06626 4.146403.03411 3.06620 2.89981 3.95571 3.34319 3.76550 3.46463 3.32709 2.581601.28546 2.73596 5.35824 4.13176 85 t - - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.033513.80839 4.53074 0.61958 0.77255 0.48576 0.95510  86 3.15726 4.672193.74475 3.46341 2.29976 3.75634 3.59289 3.17566 3.32278 2.64581 3.857783.64645 4.23235 3.64422 3.51908 3.29987 3.44402 3.04338 3.94061 1.0794486 y - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.772550.48576 0.95510  87 2.38088 4.22532 3.37373 3.12452 3.91469 3.066264.14640 3.03411 3.06620 2.89981 3.95571 3.34319 3.76550 3.46463 3.327092.58160 1.28546 2.73596 5.35824 4.13176 87 t - - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510  88 2.935674.77649 3.42005 2.94650 4.11486 3.43409 3.78227 3.65333 2.21804 3.181064.18431 3.31043 3.94105 3.00701 1.08112 3.02376 3.20684 3.38040 5.224754.05755 88 r - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.619580.77255 0.48576 0.95510  89 2.67118 4.43758 3.27171 3.10268 4.100733.12878 4.18431 3.63096 3.15473 3.26429 4.31243 3.41512 0.95588 3.545533.40634 2.83701 3.10805 3.28352 5.30361 4.22162 89 p - - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510  902.75784 4.31972 4.03530 3.69790 3.50486 3.65597 4.51118 2.05876 3.564632.20413 3.48128 3.89758 4.22110 3.92973 3.78302 3.15664 3.12163 1.110045.27795 4.00490 90 v - - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.24354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.530740.61958 0.77255 0.48576 0.95510  91 2.73092 4.35800 3.73306 3.365761.56351 3.56518 3.77172 2.75896 3.29904 2.36459 3.51384 3.56082 4.077433.56463 3.53311 2.63475 3.05442 2.60563 4.26403 2.67171 91 f - - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.63351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 92 1.11031 4.16266 3.39952 3.18951 3.99036 2.98856 4.21886 3.106493.20414 2.99084 4.03105 3.35470 3.72187 3.55158 3.45819 2.51417 2.787162.77211 5.42402 4.21744 92 a - - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.808394.53074 0.61958 0.77255 0.48576 0.95510  93 3.15726 4.67219 3.744753.46341 2.29976 3.75634 3.59289 3.17566 3.32278 2.64581 3.85778 3.646454.23235 3.64422 3.51908 3.29987 3.44402 3.04338 3.94061 1.07944 93y - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.485760.95510  94 3.08469 4.56681 4.08583 3.72691 3.11381 3.89935 4.402792.31622 3.49522 0.95845 3.15956 4.00841 4.34677 3.87896 3.68117 3.464903.38246 2.33662 4.96833 3.67637 94 l - - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.033513.80839 4.53074 0.61958 0.77255 0.48576 0.95510  95 2.83288 4.613842.86375 2.61915 3.97341 3.32530 3.77632 3.59581 2.44483 3.10440 4.135703.07769 3.87889 1.35270 2.72959 2.88411 3.13068 3.32998 5.25766 3.9476395 q - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.772550.48576 0.95510  96 2.92923 4.75570 3.06737 2.82225 3.24301 3.396091.28522 3.62204 2.66722 3.12353 4.16260 3.23362 3.94584 3.18806 2.936083.00091 3.23101 3.35853 4.70870 3.19598 96 h - - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510  97 2.380884.22532 3.37373 3.12452 3.91469 3.06626 4.14640 3.03411 3.06620 2.899813.95571 3.34319 3.76550 3.46463 3.32709 2.58160 1.28546 2.73596 5.358244.13176 97 t - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.619580.77255 0.48576 0.95510  98 2.92310 5.03735 1.01454 2.30009 4.406403.16420 3.87837 3.98050 2.95099 3.60099 4.57912 2.85261 3.81266 3.118483.45728 2.89377 3.26085 3.62704 5.58736 4.29177 98 d - - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510  992.38088 4.22532 3.37373 3.12452 3.91469 3.06626 4.14640 3.03411 3.066202.89981 3.95571 3.34319 3.76550 3.46463 3.32709 2.58160 1.28546 2.735965.35824 4.13176 99 t - - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.530740.61958 0.77255 0.48576 0.95510 100 2.98131 4.41980 4.24162 3.856203.33257 3.99480 4.59720 1.16111 3.68296 1.93351 3.27971 4.11059 4.433514.03877 3.88695 3.48513 3.28221 1.84862 5.20905 3.94078 100 i - - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510101 2.36588 4.26905 3.21789 2.89869 3.73913 3.09456 3.93689 3.159072.84145 2.61114 3.88420 3.19454 3.74532 3.23703 3.14944 1.51879 2.775352.83604 5.19347 3.84812 101 s - - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.808394.53074 0.61958 0.77255 0.48576 0.95510 102 2.98131 4.41980 4.241623.85620 3.33257 3.99480 4.59720 1.16111 3.68296 1.93351 3.27971 4.110594.43351 4.03877 3.88695 3.48513 3.28221 1.84862 5.20905 3.94078 102i - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.485760.95510 103 2.67118 4.43758 3.27171 3.10268 4.10073 3.12878 4.184313.63096 3.15473 3.26429 4.31243 3.41512 0.95588 3.54553 3.40634 2.837013.10805 3.28352 5.30361 4.22102 103 p - - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.033513.80839 4.53074 0.61958 0.77255 0.48576 0.95510 104 2.83288 4.813842.86375 2.61915 3.97341 3.32530 3.77632 3.59581 2.44483 3.10440 4.135703.07769 3.87889 1.35270 2.72959 2.88411 3.13068 3.32998 5.25766 3.94763104 q - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.772550.48576 0.95510 105 2.83288 4.81384 2.86375 2.61915 3.97341 3.325303.77632 3.59581 2.44483 3.10440 4.13570 3.07769 3.87889 1.35270 2.729592.88411 3.13068 3.32998 5.25766 3.94763 105 q - - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 106 2.757844.31972 4.03530 3.69790 3.50486 3.65597 4.51118 2.05876 3.56463 2.204133.48128 3.89758 4.22110 3.92973 3.78302 3.15664 3.12163 1.11004 5.277954.00490 106 v - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.619580.77255 0.48576 0.95510 107 2.38088 4.22532 3.37373 3.12452 3.914693.06626 4.14640 3.03411 3.06620 2.89981 3.95571 3.34319 3.76550 3.464633.32709 2.58160 1.28546 2.73596 5.35824 4.13176 107 t - - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 1082.83288 4.81384 2.86375 2.61915 3.97341 3.32530 3.77632 3.59561 2.444833.10440 4.13570 3.07769 3.87889 1.35270 2.72959 2.88411 3.13068 3.329985.25766 3.94763 108 q - - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.161462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.530740.61958 0.77255 0.48576 0.95510 109 2.38088 4.22532 3.37373 3.124523.91469 3.06626 4.14640 3.03411 3.06620 2.89981 3.95571 3.34319 3.765503.46463 3.32709 2.58160 1.28546 2.73596 5.35824 4.13176 109 t - - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510110 2.89232 4.96281 3.17183 2.66167 4.34568 3.49482 3.58934 3.734781.33144 3.24825 4.14154 3.08520 3.92108 2.74582 2.04245 2.91186 3.100003.42808 5.34210 4.13228 110 k - - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.808394.53074 0.61958 0.77255 0.48576 0.95510 111 2.28599 4.20474 3.153132.96740 4.01316 2.93592 4.07832 3.52777 3.03676 3.25353 4.18606 3.201533.66974 3.39147 3.32378 1.19199 2.76159 3.07699 5.39114 4.09669 111s - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.485760.95510 112 3.15726 4.67219 3.74475 3.46341 2.29976 3.75634 3.592893.17566 3.32278 2.64581 3.85778 3.64645 4.23235 3.64422 3.51908 3.299873.44402 3.04338 3.94061 1.07944 112 y - - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.033513.80839 4.53074 0.61958 0.77255 0.48576 0.95510 113 2.83288 4.813842.86375 2.61915 3.97341 3.32530 3.77632 3.59581 2.44483 3.10440 4.135703.07769 3.87889 1.35270 2.72959 2.88411 3.13068 3.32998 5.25766 3.94763113 q - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.03351 3.80639 4.53074 0.61958 0.772550.48576 0.95510 114 3.08469 4.56681 4.08583 3.72691 3.11381 3.899354.40279 2.31622 3.49522 0.95845 3.15956 4.00841 4.34677 3.87896 3.681173.46490 3.38246 2.33662 4.96833 3.67637 114 l - - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 115 2.285994.20474 3.15313 2.96740 4.01316 2.93592 4.07832 3.52777 3.03676 3.253534.18606 3.20153 3.66974 3.39147 3.32378 1.19199 2.76159 3.07699 5.391144.09669 115 s - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.619580.77255 0.48576 0.95510 116 2.91557 4.87384 3.08714 2.72252 4.245573.41039 3.72746 3.67115 1.12970 3.25103 4.21733 3.14931 3.91643 2.923632.42483 2.96260 3.17436 3.38305 5.32773 4.13178 116 k - - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.16146 2.89601 2.37887 2.77519 2.98518 4.584773.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 1172.58929 4.36940 3.24976 3.14188 4.28221 0.83523 4.26473 3.86748 3.314253.52541 4.50578 3.41443 3.75846 3.64375 3.55601 2.75963 3.06560 3.408325.39294 4.38245 117 g - - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.530740.61958 0.77255 0.48576 0.95510 118 2.92923 4.75570 3.06737 2.822253.24301 3.39609 1.28522 3.62204 2.66722 3.12353 4.16260 3.23362 3.945843.16806 2.93608 3.00091 3.23101 3.35853 4.70870 3.19598 118 h - - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510119 2.38088 4.22532 3.37373 3.12452 3.91469 3.06626 4.14640 3.034113.06620 2.89981 3.95571 3.34319 3.76550 3.46463 3.32709 2.58160 1.285462.73596 5.35824 4.13176 119 t - - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.808394.53074 0.61958 0.77255 0.48576 0.95510 120 2.83288 4.81384 2.863752.61915 3.97341 3.32530 3.77632 3.59581 2.44483 3.10440 4.13570 3.077693.87889 1.35270 2.72959 2.88411 3.13068 3.32998 5.25766 3.94763 120q - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.485760.95510 121 2.24451 4.20330 3.08217 2.84301 4.11231 2.92809 3.999363.56680 2.92262 3.25198 4.11942 3.11757 3.03611 3.26149 3.24979 1.393312.70133 3.08921 5.45557 4.18647 121 s - - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.033513.80839 4.53074 0.61958 0.77255 0.48576 0.95510 122 3.17889 4.600084.15032 3.85387 1.12282 3.88119 3.85245 2.71188 3.76280 2.09750 3.452513.94779 4.34224 3.94887 3.89833 3.45132 3.48052 2.69761 4.12292 2.49894122 f - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.772550.48576 0.95510 123 2.38088 4.22532 3.37373 3.12452 3.91469 3.066264.14640 3.03411 3.06620 2.89981 3.95571 3.34319 3.76550 3.46463 3.327092.58160 1.28546 2.73596 5.35824 4.13176 123 t - - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.24354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 124 2.915574.87384 3.08714 2.72252 4.24557 3.41039 3.72746 3.67115 1.12970 3.251034.21733 3.14931 3.91643 2.92363 2.42483 2.96260 3.17436 3.38305 5.327734.13178 124 k - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.619580.77255 0.48576 0.95510 125 2.28599 4.20474 3.15313 2.96740 4.013162.93592 4.07832 3.52777 3.03676 3.25353 4.18606 3.20153 3.66974 3.391473.32378 1.19199 2.76159 3.07699 5.39114 4.09669 125 s - - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 1262.75784 4.31972 4.03530 3.69790 3.50486 3.65597 4.51118 2.05876 3.564632.20413 3.48128 3.89758 4.22110 3.92973 3.78302 3.15664 3.12163 1.110045.27795 4.00490 126 v - - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.530740.61958 0.77255 0.48576 0.95510 127 2.28599 4.20474 3.15313 2.967404.01316 2.93592 4.07832 3.52777 3.03676 3.25353 4.18606 3.20153 3.669743.39147 3.32378 1.19199 2.76159 3.07699 5.39114 4.09669 127 s - - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510128 1.11031 4.16266 3.39952 3.18951 3.90036 2.98856 4.21886 3.106493.20414 2.99084 4.03105 3.35470 3.72187 3.55158 3.45819 2.51417 2.787162.77211 5.42402 4.21744 128 a - - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.808394.53074 0.61958 0.77255 0.48576 0.95510 129 2.91557 4.87384 3.087142.72252 4.24557 3.41039 3.72746 3.67115 1.12970 3.25103 4.21733 3.149313.91643 2.92363 2.42483 2.96260 3.17436 3.38305 5.32773 4.13178 129k - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.485760.95510 130 3.15726 4.67219 3.74475 3.46341 2.29976 3.75634 3.592893.17566 3.32278 2.64581 3.85778 3.64645 4.23235 3.64422 3.51908 3.299873.44402 3.04338 3.94061 1.07944 130 y - - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.033513.80839 4.53074 0.61958 0.77255 0.48576 0.95510 131 2.28599 4.204743.15313 2.96740 4.01316 2.93592 4.07832 3.52777 3.03676 3.25353 4.186063.20153 3.66974 3.39147 3.32378 1.19199 2.76159 3.07699 5.39114 4.09669131 s - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.772550.48576 0.95510 132 2.75784 4.31972 4.03530 3.69790 3.50486 3.655974.51118 2.05876 3.56463 2.20413 3.48128 3.89758 4.22110 3.92973 3.783023.15664 3.12163 1.11004 5.27795 4.00490 132 v - - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89601 2.37887 2.77519 2.98518 4.58477 3.615030.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 133 2.589294.36940 3.24976 3.14188 4.28221 0.83523 4.26473 3.86748 3.31425 3.529414.50578 3.41443 3.75846 3.64375 3.55601 2.75963 3.06560 3.40832 5.392944.38245 133 g - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.619580.77255 0.48576 0.95510 134 2.58929 4.36940 3.24976 3.14188 4.282210.83523 4.26473 3.86748 3.31425 3.52941 4.50578 3.41443 3.75846 3.643753.55601 2.75963 3.06560 3.40832 5.39294 4.38245 134 g - - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 1352.28599 4.20474 3.15313 2.96740 4.01316 2.93592 4.07832 3.52777 3.036763.25353 4.18606 3.20153 3.66974 3.39147 3.32378 1.19199 2.76159 3.076995.39114 4.09669 135 s - - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.530740.61958 0.77255 0.48576 0.95510 136 2.98131 4.41980 4.24162 3.856203.33257 3.99480 4.59720 1.16111 3.68296 1.93351 3.27971 4.11059 4.433514.03877 3.88695 3.48513 3.28221 1.84862 5.20905 3.94078 136 i - - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510137 2.92310 5.03735 1.01454 2.30009 4.40640 3.16420 3.87837 3.980502.95099 3.60099 4.57912 2.85261 3.81266 3.11848 3.45728 2.89377 3.260853.62704 5.58736 4.29177 137 d - - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.808394.53074 0.61958 0.77255 0.48576 0.95510 138 2.98131 4.41980 4.241623.85620 3.33257 3.99480 4.59720 1.16111 3.68296 1.93351 3.27971 4.110594.43351 4.03877 3.88695 3.48513 3.28221 1.84862 5.20905 3.94078 138i - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.485760.95510 139 2.75784 4.31972 4.03530 3.69790 3.50486 3.65597 4.511182.05876 3.56463 2.20413 3.48128 3.89758 4.22110 3.92973 3.78302 3.156643.12163 1.11004 5.27795 4.00490 139 v - - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.033513.80839 4.53074 0.61958 0.77255 0.48576 0.95510 140 2.67266 4.664692.67738 2.55713 4.01837 3.14279 3.87335 3.71632 2.77246 3.37097 4.327721.27686 3.79220 3.14721 3.12388 2.74309 3.05315 3.34503 5.31189 3.97225140 n - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.24354 2.67741 2.69355 4.24690 2.90147 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.772550.48576 0.95510 141 2.96177 4.37429 4.41281 3.94113 3.36272 4.136524.69082 1.27201 3.78761 1.89165 3.22369 4.19385 4.51411 4.09740 4.007763.53044 3.24517 1.61177 5.29720 4.06537 141 i - - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 142 2.285994.20474 3.15313 2.96740 4.01316 2.93592 4.07832 3.52777 3.03676 3.253534.18606 3.20153 3.66974 3.39147 3.32378 1.19199 2.76159 3.07699 5%391144.09669 142 s - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.619580.77255 0.48576 0.95510 143 2.28599 4.20474 3.15313 2.96740 4.613162.93592 4.07832 3.52777 3.03676 3.25353 4.18606 3.20153 3.66974 3.391473.32378 1.19199 2.76159 3.07699 5.39114 4.09669 143 s - - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 1442.92310 5.03735 1.01454 2.30009 4.40640 3.16420 3.87837 3.98050 2.950993.60099 4.57912 2.85261 3.81266 3.11848 3.45728 2.89377 3.26085 3.627045.58736 4.29177 144 d - - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.530740.61958 0.77255 0.48576 0.95510 145 2.98131 4.41980 4.24162 3.856203.33257 3.99480 4.59720 1.16111 3.68296 1.93351 3.27971 4.11059 4.433514.03877 3.88695 3.48513 3.28221 1.84862 5.20905 3.94078 145 i - - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510146 2.38088 4.22532 3.37373 3.12452 3.91469 3.06626 4.14640 3.034113.06620 2.89981 3.95571 3.34319 3.76550 3.46463 3.32709 2.58160 1.285462.73596 5.35824 4.13176 146 t - - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.808394.53074 0.61958 0.77255 0.48576 0.95510 147 2.75784 4.31972 4.035303.69790 3.50486 3.65597 4.51118 2.05876 3.56463 2.20413 3.48128 3.897584.22110 3.92973 3.78302 3.15664 3.12163 1.11004 5.27795 4.00490 147v - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.485760.95510 148 2.58929 4.36940 3.24976 3.14188 4.28221 0.83523 4.264733.86748 3.31425 3.52941 4.50578 3.41443 3.75846 3.64375 3.55601 2.759633.06560 3.40832 5.39294 4.38245 148 g - - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.033513.80839 4.53074 0.61958 0.77255 0.48576 0.95510 149 3.17889 4.600084.15032 3.85387 1.12282 3.88119 3.85245 2.71188 3.76280 2.09750 3.452513.94779 4.34224 3.94887 3.89833 3.45132 3.48052 2.69761 4.12292 2.49894149 f - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.772550.48576 0.95510 150 2.28599 4.20474 3.15313 2.96740 4.01316 2.935924.07832 3.52777 3.03676 3.25353 4.18606 3.20153 3.66974 3.39147 3.323781.19199 2.76159 3.07699 5.39114 4.09669 150 s - - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 151 2.285994.20474 3.15313 2.96740 4.01316 2.93592 4.07832 3.52777 3.03676 3.253534.18606 3.20153 3.66974 3.39147 3.32378 1.19199 2.76159 3.07699 5.391144.09669 151 s - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.619580.77255 0.48576 0.95510 152 2.38088 4.22532 3.37373 3.12452 3.914693.06626 4.14640 3.03411 3.06620 2.89981 3.95571 3.34319 3.76550 3.464633.32709 2.58160 1.28546 2.73596 5.35824 4.13176 152 t - - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 1532.89992 4.99376 2.46525 1.12027 4.33240 3.23467 3.81790 3.77361 2.690523.40180 4.38024 2.90101 3.83668 3.03593 3.09149 2.88487 3.20420 3.464695.51622 4.24017 153 e - - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.530740.61958 0.77255 0.48576 0.95510 154 1.11031 4.16266 3.39952 3.189513.99036 2.98856 4.21886 3.10649 3.20414 2.99084 4.03105 3.35470 3.721873.55158 3.45819 2.51417 2.78716 2.77211 5.42402 4.21744 154 a - - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510155 3.23357 4.63032 3.99599 3.68726 2.60690 3.66065 3.82592 3.255713.41132 2.66106 3.89235 3.84955 4.17341 3.80241 3.55053 3.43778 3.531063.14428 1.02517 2.61260 155 w - - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.808394.53074 0.61958 0.77255 0.48576 0.95510 156 2.28599 4.20474 3.153132.96740 4.01316 2.93592 4.07832 3.52777 3.03676 3.25353 4.18606 3.201533.66974 3.39147 3.32378 1.19199 2.76159 3.07699 5.39114 4.09669 156s - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.485760.95510 157 2.38088 4.22532 3.37373 3.12452 3.91469 3.06626 4.146403.03411 3.06620 2.89981 3.95571 3.34319 3.76550 3.46463 3.32709 2.581601.28546 2.73596 5.35824 4.13176 157 t - - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.901472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.033513.80839 4.53074 0.61958 0.77255 0.48576 0.95510 158 2.49754 4.546312.69929 2.50989 4.12129 3.09926 3.61647 3.55010 2.66261 3.25288 4.149001.68050 3.73627 3.03747 3.03371 2.58350 2.55829 3.16634 5.44237 4.09318158 n - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.772550.48576 0.95510 159 2.83288 4.81384 2.86375 2.61915 3.97341 3.325303.77632 3.59581 2.44483 3.10440 4.13570 3.07769 3.87889 1.35270 2.729592.88411 3.13068 3.32998 5.25766 3.94763 159 q - - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 160 2.380884.22532 3.37373 3.12452 3.91469 3.06626 4.14640 3.03411 3.06620 2.899813.95571 3.34319 3.76550 3.46463 3.32709 2.58160 1.28546 2.73596 5.358244.13176 160 t - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.619580.77255 0.48576 0.95510 161 3.17889 4.60008 4.15032 3.85387 1.122823.88119 3.85245 2.71188 3.76280 2.09750 3.45251 3.94779 4.34224 3.948873.89833 3.45132 3.48052 2.69761 4.12292 2.49894 161 f - - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 1622.38088 4.22532 3.37373 3.12452 3.91469 3.06626 4.14640 3.03411 3.066202.89981 3.95571 3.34319 3.76550 3.46463 3.32709 2.58160 1.28546 2.735965.35824 4.13176 162 t - - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.530740.61958 0.77255 0.48576 0.95510 163 2.83288 4.81384 2.86375 2.619153.97341 3.32530 3.77632 3.59581 2.44483 3.10440 4.13570 3.07769 3.878891.35270 2.72959 2.88411 3.13068 3.32998 5.25766 3.94763 163 q - - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510164 2.28599 4.20474 3.15313 2.96740 4.01316 2.93592 4.07832 3.527773.03676 3.25353 4.18606 3.20153 3.66974 3.39147 3.32378 1.19199 2.761593.07699 5.39114 4.09669 164 s - - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.808394.53074 0.61958 0.77255 0.48576 0.95510 165 2.38088 4.22532 3.373733.12452 3.91469 3.06626 4.14640 3.03411 3.06620 2.89981 3.95571 3.343193.76550 3.46463 3.32709 2.58160 1.28546 2.73596 5.35824 4.13176 165t - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.485760.95510 166 2.89992 4.99376 2.46525 1.12027 4.33240 3.23467 3.817903.77361 2.69052 3.40180 4.38024 2.90101 3.83668 3.03593 3.09149 2.889873.20420 3.46469 5.51622 4.24017 166 e - - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.033513.80839 4.53074 0.61958 0.77255 0.48576 0.95510 167 3.08469 4.566814.08583 3.72691 3.11381 3.89935 4.40279 2.31622 3.49522 0.95845 3.159564.00841 4.34677 3.87896 3.68117 3.46490 3.38246 2.33662 4.96833 3.67637167 l - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.772550.48576 0.95510 168 1.11031 4.16266 3.39952 3.18951 3.99036 2.988564.21886 3.10649 3.20414 2.99084 4.03105 3.35470 3.72187 3.55158 3.458192.51417 2.78716 2.77211 5.42402 4.21744 168 a - - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 169 2.589294.36940 3.24976 3.14188 4.28221 0.83523 4.26473 3.86748 3.31425 3.529414.50578 3.41443 3.75846 3.64375 3.55601 2.75963 3.06560 3.40832 5.392444.38245 169 g - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.619580.77255 0.48576 0.95510 170 2.67118 4.43758 3.27171 3.10268 4.100733.12878 4.18431 3.63096 3.15473 3.26429 4.31243 3.41512 0.95588 3.545533.40634 2.83701 3.10805 3.28352 5.30361 4.22102 170 p - - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 1712.58929 4.36940 3.24976 3.14188 4.28221 0.83523 4.26473 3.86748 3.314253.52941 4.50578 3.41443 3.75846 3.64375 3.55601 2.75963 3.06560 3.408325.39294 4.38245 171 g - - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.530740.61958 0.77255 0.48576 0.95510 172 2.38088 4.22532 3.37373 3.124523.91469 3.06626 4.14640 3.03411 3.06620 2.89981 3.95571 3.34319 3.765503.46463 3.32709 2.58160 1.28546 2.73596 5.35824 4.13176 172 t - - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510173 3.17889 4.60008 4.15032 3.85387 1.12282 3.88119 3.85245 2.711883.76280 2.09750 3.45251 3.94779 4.34224 3.94887 3.89833 3.45132 3.480522.69761 4.12292 2.49894 173 f - - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.808394.53074 0.61958 0.77255 0.48576 0.95510 174 3.17889 4.60008 4.150323.85387 1.12282 3.88119 3.85245 2.71188 3.76280 2.09750 3.45251 3.947794.34224 3.94887 3.89833 3.45132 3.48052 2.69761 4.12292 2.49894 174f - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90147 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.485760.95510 175 2.75784 4.31972 4.03530 3.69790 3.50486 3.65597 4.511182.05876 3.56463 2.20413 3.48128 3.89758 4.22110 3.92973 3.78302 3.156643.12163 1.11004 5.27795 4.00490 175 v - - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.033513.80839 4.53074 0.61958 0.77255 0.48576 0.95510 176 3.15726 4.672193.74475 3.46341 2.29976 3.75634 3.59289 3.17566 3.32278 2.64581 3.857783.64645 4.23235 3.64422 3.51908 3.29987 3.44402 3.04338 3.94061 1.07944176 y - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.772550.48576 0.95510 177 2.83288 4.81384 2.86375 2.61915 3.97341 3.325303.77632 3.59581 2.44483 3.10440 4.13570 3.07769 3.87889 1.35270 2.729592.88411 3.13068 3.32998 5.25766 3.94763 177 q - - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 178 2.757844.31972 4.03530 3.69790 3.50486 3.65597 4.51118 2.05876 3.56463 2.204133.48128 3.89758 4.22110 3.92973 3.78302 3.15664 3.12163 1.11004 5.277954.00490 178 v - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.619580.77255 0.48576 0.95510 179 2.75784 4.31972 4.03530 3.69790 3.504863.65597 4.51118 2.05876 3.56463 2.20413 3.48128 3.89758 4.22110 3.929733.78302 3.15664 3.12163 1.11004 5.27795 4.00490 179 v - - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 1803.17889 4.60008 4.15032 3.85387 1.12282 3.88119 3.85245 2.71188 3.762802.09750 3.45251 3.94779 4.34224 3.94887 3.89833 3.45132 3.48052 2.697614.12292 2.49894 180 f - - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.530740.61958 0.77255 0.48576 0.95510 181 2.75784 4.31972 4.03530 3.697903.50486 3.65597 4.51118 2.05876 3.56463 2.20413 3.48128 3.89758 4.221103.92973 3.78302 3.15664 3.12163 1.11004 5.27795 4.00490 181 v - - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510182 3.15726 4.67219 3.74475 3.46341 2.29976 3.75634 3.59289 3.175663.32278 2.64581 3.85778 3.64645 4.23235 3.64422 3.51908 3.29987 3.444023.04338 3.94061 1.07944 182 y - - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.808394.53074 0.61958 0.77255 0.48576 0.95510 183 1.11031 4.16266 3.399523.18951 3.99036 2.98856 4.21886 3.10649 3.20414 2.99084 4.03105 3.354703.72187 3.55158 3.45819 2.51417 2.78716 2.77211 5.42402 4.21744 183a - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90147 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.485760.95510 184 2.92923 4.75570 3.06737 2.82225 3.24301 3.39609 1.285223.62204 2.66722 3.12353 4.16260 3.23362 3.94584 3.18806 2.93608 3.000913.23101 3.35853 4.70870 3.19598 184 h - - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.033513.80839 4.53074 0.61958 0.77255 0.48576 0.95510 185 2.67266 4.664692.67738 2.55713 4.01837 3.14279 3.87335 3.71632 2.77246 3.37097 4.327721.27686 3.79220 3.14721 3.12388 2.74309 3.05315 3.34503 5.33189 3.97225185 n - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.772550.48576 0.95510 186 1.11031 4.16266 3.39952 3.18951 3.99036 2.988564.21886 3.10649 3.20414 2.99084 4.03105 3.35470 3.72187 3.55158 3.458192.51417 2.78716 2.77211 5.42402 4.21744 186 a - - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 187 2.380884.22532 3.37373 3.12452 3.91469 3.06626 4.14640 3.03411 3.06620 2.899813.95571 3.34319 3.76550 3.46463 3.32709 2.58160 1.28546 2.73596 5.358244.13176 187 t - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.619580.77255 0.48576 0.95510 188 2.28599 4.20474 3.15313 2.96740 4.013162.93592 4.07832 3.52777 3.03676 3.25353 4.18606 3.20153 3.66974 3.391473.32378 1.19199 2.76159 3.07699 5.39114 4.09669 188 s - 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- - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.808394.53074 0.61958 0.77255 0.48576 0.95510 192 2.83288 4.81384 2.863752.61915 3.97341 3.32530 3.77632 3.59581 2.44483 3.10440 4.13570 3.077693.87889 1.35270 2.72959 2.88411 3.13068 3.32998 5.25766 3.94763 192q - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.485760.95510 193 2.67266 4.66469 2.67738 2.55713 4.01837 3.14279 3.873353.71632 2.77246 3.37097 4.32772 1.27686 3.79220 3.14721 3.12388 2.743093.05315 3.34503 5.33189 3.97225 193 n - - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.901472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.033513.80839 4.53074 0.61958 0.77255 0.48576 0.95510 194 2.58929 4.369403.24976 3.14188 4.28221 0.83523 4.26473 3.86748 3.31425 3.52941 4.505783.41443 3.75846 3.64375 3.55601 2.75963 3.06560 3.40832 5.39294 4.38245194 g - 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- - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61543 0.03351 3.808394.53074 0.61958 0.77255 0.48576 0.95510 201 2.91557 4.87384 3.087142.72252 4.24557 3.41039 3.72746 3.67115 1.12970 3.25103 4.21733 3.149313.91643 2.92363 2.42483 2.96260 3.17436 3.38305 5.32773 4.13178 201k - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.485760.95510 202 2.38088 4.22532 3.37373 3.12452 3.51469 3.06626 4.146403.03411 3.06620 2.89981 3.95571 3.34319 3.76550 3.46463 3.32709 2.581601.28546 2.73596 5.35824 4.13176 202 t - - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.25354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.033513.80839 4.53074 0.61958 0.77255 0.48576 0.95510 203 2.83288 4.813842.86375 2.61915 3.97341 3.32530 3.77632 3.59581 2.44483 3.10440 4.135703.07769 3.87889 1.35270 2.72959 2.88411 3.13068 3.32998 5.25766 3.94763203 q - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.772550.48576 0.95510 204 2.83288 4.81384 2.86375 2.61915 3.97341 3.325303.77632 3.59581 2.44483 3.10440 4.13570 3.07769 3.87889 1.35270 2.729592.88411 3.13068 3.32998 5.25766 3.94763 204 q - - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 205 2.757844.31972 4.03530 3.69790 3.50486 3.65597 4.51118 2.05876 3.56463 2.204133.48128 3.89758 4.22110 3.92973 3.78302 3.15664 3.12163 1.11004 5.277954.00490 205 v - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 238518 4.58477 3.61503 0.03351 3.80839 4.53074 0.619580.77255 0.48576 0.95510 206 2.67266 4.66469 2.67738 2.55713 4.018373.14279 3.87335 3.71632 2.77246 3.37097 4.32772 1.27686 3.79220 3.147213.12388 2.74309 3.05315 3.34503 5.33189 3.97225 206 n - - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 2072.28599 4.20474 3.15313 2.96740 4.01316 2.93592 4.07832 3.52777 3.036763.25353 4.18606 3.20153 3.66974 3.39147 3.32378 1.19199 2.76159 3.076995.39114 4.09669 207 s - - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.530740.61958 0.77255 0.48576 0.95510 208 2.93567 4.77649 3.42005 2.946504.11486 3.43409 3.78227 3.65333 2.21804 3.18106 4.18431 3.31043 3.941053.00701 1.08112 3.02376 3.20684 3.38040 5.22475 4.05755 208 r - - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510209 3.08469 4.56681 4.08583 3.72691 3.11381 3.89935 4.40279 2.316223.49522 0.95845 3.15956 4.00841 4.34677 3.87896 3.68117 3.46490 3.382462.33662 4.96833 3.67637 209 l - - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.63351 3.808394.53074 0.61958 0.77255 0.48576 0.95510 210 2.92310 5.03735 1.014542.30009 4.40640 3.16420 3.87837 3.98050 2.95099 3.60099 4.57912 2.852613.81266 3.11848 3.45728 2.89377 3.26085 3.62704 5.58736 4.29177 210d - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.485760.95510 211 3.08469 4.56681 4.08583 3.72691 3.11381 3.69935 4.402792.31622 3.49522 0.95845 3.15956 4.00841 4.34677 3.87896 3.68117 3.464903.38246 2.33662 4.96833 3.67637 211 l - - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.033513.80839 4.53074 0.61958 0.77255 0.48576 0.95510 212 3.15726 4.672193.74475 3.46341 2.29976 3.75634 3.59289 3.17566 3.32278 2.64581 3.857783.64645 4.23235 3.64422 3.51908 3.29987 3.44402 3.04338 3.94061 1.07944212 y - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.772550.48576 0.95510 213 3.15726 4.67219 3.74475 3.46341 2.29976 3.756343.59289 3.17566 3.32278 2.64581 3.85778 3.64645 4.23235 3.64422 3.519083.29987 3.44402 3.04338 3.94061 1.07544 213 y - - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 214 3.084694.56681 4.08583 3.72691 3.11381 3.89935 4.40279 2.31622 3.49522 0.958453.15956 4.60841 4.34677 3.87896 3.68117 3.46490 3.38246 2.33662 4.968333.67637 214 l - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.619580.77255 0.48576 0.95510 215 2.28599 4.20474 3.15313 2.96740 4.013162.93592 4.07832 3.52777 3.03676 3.25353 4.18606 3.20153 3.66974 3.391473.32378 1.19199 2.76159 3.07699 5.39114 4.09669 215 s - - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 2161.11031 4.16266 3.39952 3.18951 3.99036 2.98856 4.21886 3.10649 3.204142.99084 4.03105 3.35470 3.72187 3.55158 3.45819 2.51417 2.78716 2.772115.42402 4.21744 216 a - - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.530740.61958 0.77255 0.48576 0.95510 217 2.98131 4.41980 4.24162 3.856203.33257 3.99480 4.59720 1.16111 3.68296 1.93351 3.27971 4.11059 4.433514.03877 3.88695 3.48513 3.28221 1.84862 5.20905 3.94078 217 i - - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510218 2.38088 4.22532 3.37373 3.12452 3.91469 3.06626 4.14640 3.034113.06620 2.89981 3.95571 3.34319 3.76550 3.46463 3.32709 2.58160 1.285462.73596 5.35824 4.13176 218 t - - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.808394.53074 0.61958 0.77255 0.48576 0.95510 219 2.61338 4.63513 2.989822.49568 3.86125 3.37483 3.58066 3.33848 2.28320 2.95092 3.79875 2.969403.78724 1.96490 2.62957 2.67100 2.84872 3.04772 5.06862 3.82222 219q - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.485760.95510 220 2.92310 5.03735 1.01454 2.30009 4.40640 3.16420 3.878373.98050 2.95099 3.60099 4.57912 2.85261 3.81266 3.11848 3.45726 2.893773.26085 3.62704 5.58736 4.29177 220 d - - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.033513.80839 4.53074 0.61958 0.77255 0.48576 0.95510 221 2.93567 4.776493.42005 2.94650 4.11486 3.43409 3.78227 3.65333 2.21804 3.18106 4.184313.31043 3.94105 3.00701 1.08112 3.02376 3.20684 3.38040 5.22475 4.05755221 r - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.772550.48576 0.95510 222 2.38088 4.22532 3.37373 3.12452 3.91469 3.066264.14640 3.03411 3.06620 2.89981 3.95571 3.34319 3.76550 3.46463 3.327092.58160 1.28546 2.73596 5.35824 4.13176 222 t - - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 223 2.757844.31972 4.03530 3.69790 3.50486 3.65597 4.51118 2.05876 3.56463 2.204133.48128 3.89758 4.22110 3.92973 3.78302 3.15664 3.12163 1.11004 5.277954.00490 223 v - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.619580.77255 0.48576 0.95510 224 2.98131 4.41980 4.2416Z 3.85620 3.332573.99480 4.59720 1.16111 3.68296 1.93351 3.27971 4.11059 4.43351 4.038773.88695 3.48513 3.28221 1.84862 5.20905 3.94078 224 i - - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90147 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 2252.75784 4.31972 4.03530 3.69790 3.50486 3.65597 4.51118 2.05876 3.564632.20413 3.48128 3.89758 4.22110 3.92973 3.78302 3.15664 3.12163 1.110045.27795 4.00490 225 v - - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.530740.61958 0.77255 0.48576 0.95510 226 2.89992 4.99376 2.46525 1.120274.33240 3.23467 3.81790 3.77361 2.69052 3.40180 4.38024 2.90101 3.836683.03593 3.09149 2.88987 3.20420 3.46469 5.51622 4.24017 226 e - - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510227 2.28599 4.20474 3.15313 2.96740 4.01316 2.93592 4.07832 3.527773.03676 3.25353 4.18606 3.20153 3.66974 3.39147 3.32378 1.19199 2.761593.07699 5.39114 4.09669 227 s - - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.808394.53074 0.61958 0.77255 0.48576 0.95510 228 2.28599 4.20474 3.153132.96740 4.01316 2.93592 4.07832 3.52777 3.03676 3.25353 4.18606 3.201533.66974 3.39147 3.32378 1.19199 2.76159 3.07699 5.39114 4.09669 228s - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.485760.95510 229 2.71151 4.86762 2.55085 2.36054 4.22135 3.22801 3.694543.73053 2.23879 3.33117 4.21189 1.69444 3.79743 2.88684 2.79192 2.725233.01237 3.37329 5.44965 4.09171 229 n - - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.033513.80839 4.53074 0.61958 0.77255 0.48576 0.95510 230 1.11031 4.162663.39952 3.18951 3.99036 2.98856 4.21886 3.10649 3.20414 2.99084 4.031053.35470 3.72187 3.55158 3.45819 2.51417 2.78716 2.77211 5.42402 4.21744230 a - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.772550.48576 0.95510 231 2.98131 4.41980 4.24162 3.85620 3.33257 3.994804.59720 1.16111 3.68296 1.93351 3.27971 4.11059 4.43351 4.03877 3.886953.48513 3.28221 1.84862 5.20905 3.94078 231 i - - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90147 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 232 2.432424.94379 1.41602 2.20498 4.38970 3.12406 3.76776 3.77053 2.76895 3.453644.36242 2.74764 3.75771 2.96307 3.29061 2.69377 3.04302 3.40240 5.672164.25963 232 d - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.619580.77255 0.48576 0.95510 233 2.67118 4.43758 3.27171 3.10268 4.100733.12878 4.18431 3.63096 3.15473 3.26429 4.31243 3.41512 0.95588 3.545533.40634 2.83701 3.10805 3.28352 5.30361 4.22102 233 p - - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 2343.08469 4.56681 4.08583 3.72691 3.11381 3.89935 4.40279 2.31622 3.495220.95845 3.15956 4.00841 4.34677 3.87896 3.68117 3.46490 3.38246 2.336624.96833 3.67637 234 l - - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.530740.61958 0.77255 0.48576 0.95510 235 2.92310 5.03735 1.01454 2.300094.40640 3.16420 3.87837 3.98050 2.95099 3.60099 4.57912 2.85261 3.812663.11848 3.45728 2.89377 3.26085 3.62704 5.58736 4.29177 235 d - - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510236 2.85644 4.50201 3.66038 3.16223 2.82982 3.60762 3.68523 3.063932.72567 2.60253 3.65678 3.45451 4.04446 3.26564 2.56136 3.03906 3.109122.88190 1.77632 2.85170 236 w - - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.808394.53074 0.61958 0.77255 0.48576 0.95510 237 2.92310 5.03735 1.014542.30009 4.40640 3.16420 3.87837 3.98050 2.95099 3.60099 4.57912 2.852613.81266 3.11848 3.45728 2.89377 3.26085 3.62704 5.58736 4.29177 237d - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.485760.95510 238 2.38088 4.22532 3.37373 3.12452 3.91469 3.06626 4.146403.03411 3.06620 2.89981 3.95571 3.34319 3.76550 3.46463 3.32709 2.581601.28546 2.73596 5.35824 4.13176 238 t - - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.033513.80839 4.53074 0.61958 0.77255 0.48576 0.95510 239 2.75784 4.319724.03530 3.69790 3.50486 3.65597 4.51118 2.05876 3.56463 2.20413 3.481283.89758 4.22110 3.92973 3.78302 3.15664 3.12163 1.11004 5.27795 4.00490239 v - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.16146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.772550.48576 0.95510 240 2.83288 4.81384 2.86375 2.61915 3.97341 3.325303.77632 3.59581 2.44483 3.10440 4.13570 3.07769 3.87889 1.35270 2.729592.88411 3.13068 3.32998 5.25766 3.94763 240 q - - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.24354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 241 2.935674.77649 3.42005 2.94650 4.11486 3.43409 3.78227 3.65333 2.21804 3.181064.18431 3.31043 3.94105 3.00701 1.08112 3.02376 3.20684 3.38040 5.224754.05755 241 r - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.619580.77255 0.48576 0.95510 242 2.67266 4.66469 2.67738 2.55713 4.018373.14279 3.87335 3.71632 2.77246 3.37097 4.32772 1.27686 3.79220 3.147213.12388 2.74309 3.05315 3.34503 5.33189 3.97225 242 n - - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 2432.75784 4.31972 4.03530 3.69790 3.50486 3.65597 4.51118 2.05876 3.564632.20413 3.48128 3.89758 4.22110 3.92973 3.78302 3.15664 3.12163 1.110045.27795 4.00490 243 v - - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.530740.61958 0.77255 0.48576 0.95510 244 3.08469 4.56681 4.08583 3.726913.11381 3.89935 4.40279 2.31622 3.49522 0.95845 3.15956 4.00841 4.346773.87896 3.68117 3.46490 3.38246 2.33662 4.96833 3.67637 244 l - - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89601 2.37687 2.77519 2.985184.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510245 3.01012 4.41428 4.41093 3.89648 3.23739 4.19710 4.60930 1.330473.71957 1.73611 2.77119 4.17860 4.51388 4.01448 3.93268 3.56091 3.266881.83382 5.18268 3.98317 245 i - - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.808394.53074 0.61958 0.77255 0.48576 0.95510 246 2.79822 4.99164 2.589172.18348 4.25929 3.31397 3.64736 3.71794 2.30881 3.26588 4.16696 2.869133.83263 1.72335 2.64609 2.78808 3.05306 3.39930 5.44088 4.11133 246q - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.485760.95510 247 2.67266 4.64469 2.67738 2.55713 4.01837 3.14279 3.873353.71632 2.77246 3.37097 4.32772 1.27686 3.79220 3.14721 3.12388 2.743093.05315 3.34503 5.33189 3.97225 247 n - - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.033513.80839 4.53074 0.61958 0.77255 0.48576 0.95510 248 3.15726 4.672193.74475 3.46341 2.29976 3.75634 3.59289 3.17566 3.32278 2.64581 3.857783.64645 4.23235 3.64422 3.51908 3.29987 3.44402 3.04338 3.94061 1.07944248 y - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.772550.48576 0.95510 249 2.67266 4.66469 2.67738 2.55713 4.01837 3.142793.87335 3.71632 2.77246 3.37097 4.32772 1.27686 3.79220 3.14721 3.123882.74309 3.05315 3.34503 5.33189 3.97225 249 n - - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 250 2.574394.57351 2.83143 2.58496 4.01396 3.18385 3.80153 3.50981 2.59095 3.104654.04186 3.02031 1.77449 2.65661 2.92673 2.66527 2.92228 3.17417 5.325414.02468 250 p - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.619580.77255 0.48576 0.95510 251 1.29653 4.08489 3.30251 3.06256 4.069912.88906 4.12659 3.31602 3.09903 3.14997 4.05671 3.22665 3.63269 3.423603.38369 2.13641 2.65470 2.88235 5.47235 4.23432 251 a - - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 2522.28599 4.20474 3.15313 2.96740 4.01316 2.93592 4.07832 3.52777 3.036763.25353 4.18606 3.20153 3.66974 3.39147 3.32378 1.19199 2.76159 3.076995.39114 4.09669 252 s - - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90147 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.530740.61958 0.77255 0.48576 0.95510 253 2.67266 4.66469 2.67738 2.557134.01837 3.14279 3.87335 3.71632 2.77246 3.37097 4.32772 1.27686 3.792203.14721 3.12388 2.74309 3.05315 3.34503 5.33189 3.97225 253 n - - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90147 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510254 2.28599 4.20474 3.15313 2.96740 4.01316 2.93592 4.07832 3.527773.03676 3.25353 4.18606 3.20153 3.66974 3.39147 3.32378 1.19199 2.761593.07699 5.39114 4.09669 254 s - - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.808394.53074 0.61958 0.77255 0.48576 0.95510 255 2.58929 4.36940 3.249763.14188 4.28221 0.83523 4.26473 3.86748 3.31425 3.52941 4.50578 3.414433.75846 3.64375 3.55601 2.75463 3.06560 3.40832 5.39294 4.38245 255g - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.485760.95510 256 2.92923 4.75570 3.06737 2.82225 3.24301 3.39609 1.285223.62204 2.66722 3.12353 4.16260 3.23362 3.94584 3.18806 2.93608 3.000913.23101 3.35853 4.70870 3.19598 256 h - - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.033513.80839 4.53074 0.61958 0.77255 0.48576 0.95510 257 3.17889 4.600084.15032 3.85387 1.12282 3.88119 3.85245 2.71188 3.76280 2.09750 3.452513.94779 4.34224 3.94887 3.89833 3.45132 3.48052 2.69761 4.12292 2.49894257 f - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.03351 3.80639 4.53074 0.61958 0.772550.48576 0.95510 258 2.28599 4.20474 3.15313 2.96740 4.01316 2.935924.07832 3.52777 3.03676 3.25353 4.18606 3.20153 3.66974 3.39147 3.323781.19199 2.76159 3.07699 5.39114 4.09669 258 s - - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 259 3.178894.60008 4.15032 3.85387 1.12282 3.88119 3.85245 2.71188 3.76280 2.097503.45251 3.94779 4.34224 3.94887 3.89833 3.45132 3.48052 2.69761 4.122922.49894 259 f - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.619580.77255 0.48576 0.95510 260 2.92310 5.03735 1.01454 2.30009 4.406403.16420 3.87837 3.98050 2.95099 3.60099 4.57912 2.85261 3.81266 3.118483.45728 2.89377 3.26085 3.62704 5.58736 4.29177 260 d - - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90147 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 2613.23357 4.63032 3.99599 3.68726 2.60690 3.66065 3.82592 3.25571 3.411322.66106 3.89235 3.84955 4.17141 3.80241 3.55053 3.43778 3.53106 3.144281.02517 2.61260 261 w - - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.530740.61958 0.77255 0.48576 0.95510 262 2.43471 4.33947 3.10763 2.650433.72256 3.22351 3.70592 3.17554 2.56941 2.85551 3.70850 3.05491 3.731482.95850 2.92934 1.78239 2.74595 2.86658 5.01126 3.78736 262 s - - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510263 1.11031 4.16266 3.39952 3.18951 3.99036 2.98856 4.21886 3.106493.20414 2.99084 4.03105 3.35470 3.72187 3.55158 3.45819 2.51417 2.787162.77211 5.42402 4.21744 263 a - - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.808394.53074 0.61958 0.77255 0.48576 0.95510 264 3.15726 4.67219 3.744753.46341 2.29976 3.75634 3.59289 3.17566 3.32278 2.64581 3.85778 3.646454.23235 3.64422 3.51908 3.29987 3.44402 3.04338 3.94061 1.07944 264y - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.485760.95510 265 2.46429 4.50277 2.76194 2.53711 4.10808 3.09914 3.814523.50193 2.65822 3.20971 4.09377 1.83311 3.72764 3.03155 3.03107 2.559472.32552 3.11459 5.42657 4.09622 265 n - - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37687 2.77519 2.98518 4.58477 3.61503 0.033513.80839 4.53074 0.61958 0.77255 0.48576 0.95510 266 2.66368 4.713141.86174 2.43871 3.87910 3.32864 3.75508 2.53007 2.66567 2.87600 3.866082.95100 3.84113 2.96441 3.11604 2.73315 2.94304 2.80811 5.30301 3.93501266 d - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.772550.48576 0.95510 267 2.57183 4.40147 3.29756 2.92021 3.58155 3.320133.92617 2.90376 2.81038 2.11477 3.65609 3.28096 1.96754 3.22756 3.117592.75095 2.91246 2.70333 5.09267 3.78923 267 p - - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.03351 3.80639 4.53074 0.61958 0.77255 0.48576 0.95510 268 2.704014.52485 3.27058 2.81601 3.16826 3.51121 2.18756 2.48108 2.61094 2.592973.62015 3.22034 3.94808 3.07183 2.91785 2.84887 2.95894 2.75950 4.699413.22516 268 h - - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.53074 0.619580.77255 0.48576 0.95510 269 2.19646 4.57897 3.20488 2.73469 4.061593.30402 3.72028 3.39767 2.27081 3.04174 3.96013 3.11981 3.82748 2.914701.70636 2.70466 2.90940 3.08482 5.28820 4.04460 269 r - - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510 2702.65927 4.55369 3.36479 2.82353 3.79413 3.43170 3.74118 3.05153 2.325802.74721 3.75184 3.20718 3.89989 2.96147 1.78252 2.75430 2.93484 2.374465.15155 3.88489 270 r - - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03351 3.80839 4.530740.61958 0.77255 0.48576 0.95510 271 2.58892 4.22418 3.49767 2.955503.00683 3.53970 3.68743 2.54874 2.83207 2.41907 3.35230 3.30705 3.914453.16371 3.13477 2.82088 2.82225 2.51305 4.49536 2.20914 271 y - - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.03351 3.80839 4.53074 0.61958 0.77255 0.48576 0.95510272 2.68027 4.58936 2.49030 1.70991 3.90297 3.05916 3.69222 3.286212.53357 2.98575 4.02875 2.86073 3.66258 2.95416 2.86307 2.74011 3.003133.02529 5.15154 3.92226 272 e - - - 2.68623 4.42138 2.77525 2.731293.46359 2.40506 3.72500 3.29359 2.67746 2.69344 4.24695 2.90352 2.737453.18152 2.89786 2.37880 2.77525 2.98524 4.58482 3.61508 0.119032.18732 * 1.27779 0.32663 0.00000 * //

Numbered Embodiments of the Disclosure

Notwithstanding the appended claims, the disclosure sets forth thefollowing numbered embodiments:

Isolated Nucleic Acids

-   -   1. An isolated nucleic acid molecule encoding an insecticidal        protein having at least about 80% sequence identity to a protein        selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO:        4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ        ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID        NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO:        30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38,        SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ        ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID        NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO:        64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, and SEQ ID NO:        72.    -   2. The isolated nucleic acid molecule of embodiment 1, wherein        said nucleic acid molecule encodes an insecticidal protein        having at least about 90% sequence identity to a protein        selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO:        4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ        ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID        NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO:        30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38,        SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ        ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID        NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO:        64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, and SEQ ID NO:        72.    -   3. The isolated nucleic acid molecule of embodiment 1, wherein        said nucleic acid molecule encodes an insecticidal protein        having at least about 95% sequence identity to a protein        selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO:        4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ        ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID        NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO:        30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38,        SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ        ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID        NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO:        64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, and SEQ ID NO:        72.    -   4. The isolated nucleic acid molecule of embodiment 1, wherein        said nucleic acid molecule encodes an insecticidal protein        having at least about 99% sequence identity to a protein        selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO:        4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ        ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID        NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO:        30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38,        SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ        ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID        NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO:        64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, and SEQ ID NO:        72.    -   5. The isolated nucleic acid molecule of embodiment 1, wherein        said nucleic acid molecule encodes an insecticidal protein        selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO:        4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ        ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID        NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO:        30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38,        SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ        ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID        NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO:        64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, and SEQ ID NO:        72.    -   6. The isolated nucleic acid molecule of embodiment 1, wherein        said nucleic acid molecule is codon optimized for expression in        a host cell of interest.    -   7. The isolated nucleic acid molecule of embodiment 1, wherein        said nucleic acid molecule is codon optimized for expression in        a plant cell.    -   8. The isolated nucleic acid molecule of embodiment 1, wherein        said nucleic acid molecule is selected from the group consisting        of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ        ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID        NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO:        25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33,        SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ        ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID        NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO:        59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67,        SEQ ID NO: 69, and SEQ ID NO: 71.    -   9. An isolated nucleic acid as presented in Table 3.    -   10. An isolated protein, polypeptide, amino acid sequence, or        variant thereof, as presented in Table 3.

Nucleotide Constructs

-   -   1. A nucleotide construct, comprising: a nucleic acid molecule        encoding an insecticidal protein having at least about 80%        sequence identity to a protein selected from the group        consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID        NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:        16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24,        SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ        ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID        NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO:        50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58,        SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ        ID NO: 68, SEQ ID NO: 70, and SEQ ID NO: 72, said nucleic acid        molecule operably linked to a heterologous regulatory element.    -   2. The nucleotide construct of embodiment 1, wherein said        nucleic acid molecule encodes an insecticidal protein having at        least about 90% sequence identity to a protein selected from the        group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,        SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ        ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID        NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO:        32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,        SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ        ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID        NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO:        66, SEQ ID NO: 68, SEQ ID NO: 70, and SEQ ID NO: 72.    -   3. The nucleotide construct of embodiment 1, wherein said        nucleic acid molecule encodes an insecticidal protein having at        least about 95% sequence identity to a protein selected from the        group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,        SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ        ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID        NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO:        32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,        SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ        ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID        NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO:        66, SEQ ID NO: 68, SEQ ID NO: 70, and SEQ ID NO: 72.    -   4. The nucleotide construct of embodiment 1, wherein said        nucleic acid molecule encodes an insecticidal protein having at        least about 99% sequence identity to a protein selected from the        group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,        SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ        ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID        NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO:        32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,        SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ        ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID        NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO:        66, SEQ ID NO: 68, SEQ ID NO: 70, and SEQ ID NO: 72.    -   5. The nucleotide construct of embodiment 1, wherein said        nucleic acid molecule encodes an insecticidal protein selected        from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID        NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:        14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22,        SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ        ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID        NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO:        48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56,        SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ        ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, and SEQ ID NO: 72.    -   6. The nucleotide construct of embodiment 1, wherein said        heterologous regulatory element is a promoter.    -   7. The nucleotide construct of embodiment 1, wherein said        nucleotide construct is contained in an expression cassette.    -   8. The nucleotide construct of embodiment 1, wherein said        heterologous regulatory element is capable of expressing the        encoded protein in a plant.    -   9. The nucleotide construct of embodiment 1, wherein said        nucleic acid molecule is codon optimized for expression in a        host cell of interest.    -   10. The nucleotide construct of embodiment 1, wherein said        nucleic acid molecule is codon optimized for expression in a        plant cell.    -   11. The nucleotide construct of embodiment 1, wherein said        nucleic acid molecule is selected from the group consisting of:        SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID        NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:        17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25,        SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ        ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID        NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO:        51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59,        SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ        ID NO: 69, and SEQ ID NO: 71.    -   12. An expression vector comprising the nucleotide construct of        embodiment 1.    -   13. A plasmid comprising the nucleotide construct of embodiment        1.    -   14. A host cell comprising the nucleotide construct of        embodiment 1.    -   15. A method of killing an insect, comprising contacting the        insect with a host cell expressing the nucleotide construct of        embodiment 1.    -   16. A prokaryotic host cell comprising the nucleotide construct        of embodiment 1.    -   17. A eukaryotic host cell comprising the nucleotide construct        of embodiment 1.    -   18. A plant cell comprising the nucleotide construct of        embodiment 1.    -   19. A monocot plant cell comprising the nucleotide construct of        embodiment 1.    -   20. A dicot plant cell comprising the nucleotide construct of        embodiment 1.    -   21. A plant stably transformed with the nucleotide construct of        embodiment 1.    -   22. A seed produced by a plant that has been stably transformed        with the nucleotide construct of embodiment 1.

Isolated Proteins

-   -   1. An isolated insecticidal protein, comprising: an amino acid        sequence with at least about 80% sequence identity to SEQ ID NO:        2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ        ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID        NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO:        28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36,        SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ        ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID        NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO:        62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70,        or SEQ ID NO: 72.    -   2. The isolated insecticidal protein of embodiment 1,        comprising: an amino acid sequence with at least about 90%        sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,        SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ        ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID        NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO:        32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,        SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ        ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID        NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO:        66, SEQ ID NO: 68, SEQ ID NO: 70, or SEQ ID NO: 72.    -   3. The isolated insecticidal protein of embodiment 1,        comprising: an amino acid sequence with at least about 95%        sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,        SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ        ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID        NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO:        32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,        SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ        ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID        NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO:        66, SEQ ID NO: 68, SEQ ID NO: 70, or SEQ ID NO: 72.    -   4. The isolated insecticidal protein of embodiment 1,        comprising: an amino acid sequence with at least about 99%        sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,        SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ        ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID        NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO:        32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,        SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ        ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID        NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO:        66, SEQ ID NO: 68, SEQ ID NO: 70, or SEQ ID NO: 72.    -   5. The isolated insecticidal protein of embodiment 1,        comprising: an amino acid sequence selected from SEQ ID NO: 2,        SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID        NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:        20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28,        SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ        ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID        NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO:        54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62,        SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, or        SEQ ID NO: 72.

Recombinant Proteins

-   -   1. A recombinant insecticidal protein, comprising: an amino acid        sequence with at least about 80% sequence identity to SEQ ID NO:        2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ        ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID        NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO:        28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36,        SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ        ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID        NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO:        62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70,        or SEQ ID NO: 72.    -   2. The recombinant insecticidal protein of embodiment 1,        comprising: an amino acid sequence with at least about 90%        sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,        SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ        ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID        NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO:        32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,        SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ        ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID        NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO:        66, SEQ ID NO: 68, SEQ ID NO: 70, or SEQ ID NO: 72.    -   3. The recombinant insecticidal protein of embodiment 1,        comprising: an amino acid sequence with at least about 95%        sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,        SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ        ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID        NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO:        32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,        SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ        ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID        NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO:        66, SEQ ID NO: 68, SEQ ID NO: 70, or SEQ ID NO: 72.    -   4. The recombinant insecticidal protein of embodiment 1,        comprising: an amino acid sequence with at least about 99%        sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,        SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ        ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID        NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO:        32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,        SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ        ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID        NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO:        66, SEQ ID NO: 68, SEQ ID NO: 70, or SEQ ID NO: 72.    -   5. The recombinant insecticidal protein of embodiment 1,        comprising: an amino acid sequence selected from SEQ ID NO: 2,        SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID        NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:        20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28,        SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ        ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID        NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO:        54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62,        SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, or        SEQ ID NO: 72.

Transgenic Plant Cells

-   -   1. A transgenic plant cell, comprising:        -   a. a DNA construct, comprising: a polynucleotide encoding a            polypeptide having at least about 80%, 81%, 82%, 83%, 84%,            85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,            97%, 98%, 99%, or greater, sequence identity to an amino            acid sequence selected from the group consisting of: SEQ ID            NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO:            10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:            18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO:            26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO:            34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO:            42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO:            50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO:            58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO:            66, SEQ ID NO: 68, SEQ ID NO: 70, and SEQ ID NO: 72; and a            heterologous regulatory sequence operably linked to the            polynucleotide.    -   2. The transgenic plant cell of embodiment 1, wherein said        polynucleotide encodes a polypeptide having an amino acid        sequence selected from the group consisting of: SEQ ID NO: 2,        SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID        NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:        20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28,        SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ        ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID        NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO:        54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62,        SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, and        SEQ ID NO: 72.    -   3. The transgenic plant cell of embodiment 1, wherein said        heterologous regulatory element is a promoter.    -   4. The transgenic plant cell of embodiment 1, wherein said cell        is from a monocot species.    -   5. The transgenic plant cell of embodiment 1, wherein said cell        is from corn, wheat, oat, or rice.    -   6. The transgenic plant cell of embodiment 1, wherein said cell        is from a dicot species.    -   7. The transgenic plant cell of embodiment 1, wherein said cell        is from cotton, potato, or soybean.    -   8. The transgenic plant cell of embodiment 1, wherein said cell        is from an agricultural row crop species.

Transgenic Plant

-   -   1. A transgenic plant stably transformed with a DNA construct,        comprising:        -   a. a polynucleotide encoding a polypeptide having at least            about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,            91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater,            sequence identity to an amino acid sequence selected from            the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID            NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID            NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID            NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID            NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID            NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID            NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID            NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID            NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID            NO: 70, and SEQ ID NO: 72; and        -   b. a heterologous regulatory sequence operable linked to the            polynucleotide.    -   2. The transgenic plant of embodiment 1, wherein said        polynucleotide encodes a polypeptide having an amino acid        sequence selected from the group consisting of: SEQ ID NO: 2,        SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID        NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:        20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28,        SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ        ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID        NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO:        54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62,        SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, and        SEQ ID NO: 72.    -   3. The transgenic plant of embodiment 1, wherein said        heterologous regulatory element is a promoter.    -   4. The transgenic plant of embodiment 1, wherein said plant is a        monocot species.    -   5. The transgenic plant of embodiment 1, wherein said plant is        corn, wheat, oat, or rice.    -   6. The transgenic plant of embodiment 1, wherein said plant is a        dicot species.    -   7. The transgenic plant of embodiment 1, wherein said plant is        cotton, potato, or soybean.    -   8. The transgenic plant of embodiment 1, wherein said plant is        from an agricultural row crop species.    -   9. A seed produced by the plant of embodiment 1.    -   10. A progeny plant produced from the plant of embodiment 1.    -   11. The transgenic plant of embodiment 1, further comprising: a        DNA construct comprising a polynucleotide encoding a Monalysin        protein, Pseudomonas insecticidal protein, Cry protein, Cyt        protein, vegetative insecticidal protein, toxin complex protein,        and any combination thereof.    -   12. A method of killing a target pest, comprising: providing the        transgenic plant of embodiment 1 to an area, wherein said target        pest is exposed to the transgenic plant.    -   13. A method of killing a target pest, comprising: providing the        transgenic plant of embodiment 1 to an area, wherein said target        pest feeds on the transgenic plant.    -   14. A method of killing a target pest that is resistant to a        pesticidal protein, comprising: providing the transgenic plant        of embodiment 1 to an area, wherein said target pest is exposed        to the transgenic plant, and wherein the target pest is        resistant to at least one of a Monalysin protein, Pseudomonas        insecticidal protein, Cry protein, Cyt protein, vegetative        insecticidal protein, toxin complex protein, and any combination        thereof.    -   15. A method of killing a target pest that is resistant to a        pesticidal protein, comprising: providing the transgenic plant        of embodiment 1 to an area, wherein said target pest feeds on        the transgenic plant, and wherein the target pest is resistant        to at least one of a Monalysin protein, Pseudomonas insecticidal        protein, Cry protein, Cyt protein, vegetative insecticidal        protein, toxin complex protein, and any combination thereof.    -   16. A method of killing a target pest, comprising: providing the        transgenic plant of embodiment 1 to an area, wherein said target        pest is exposed to the transgenic plant and said target pest is        a member of the Order Coleoptera, Diptera, Hymenoptera,        Lepidoptera, Hemiptera, Orthroptera, Thysanoptera, or        Dermaptera.

Agricultural Compositions

-   -   1. An insecticidal composition, comprising:        -   a. an isolated insecticidal protein having an amino acid            sequence with at least about 80%, 81%, 82%, 83%, 84%, 85%,            86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,            98%, 99%, or greater, sequence identity to an amino acid            sequence selected from the group consisting of: SEQ ID NO:            2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,            SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18,            SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26,            SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34,            SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42,            SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50,            SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58,            SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66,            SEQ ID NO: 68, SEQ ID NO: 70, and SEQ ID NO: 72; and        -   b. an agriculturally acceptable carrier.    -   2. The insecticidal composition of embodiment 1, wherein the        isolated insecticidal protein has an amino acid sequence        selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO:        4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ        ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID        NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO:        30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38,        SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ        ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID        NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO:        64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, and SEQ ID NO:        72.    -   3. The insecticidal composition of embodiment 1, wherein said        isolated insecticidal protein is present in an insecticidally        effective amount.    -   4. The insecticidal composition of embodiment 1, wherein said        agriculturally acceptable carrier is at least one selected from        the group consisting of: adjuvants, inert components,        dispersants, surfactants, sticking agents, tackifiers, binders,        natural or regenerated mineral substances, solvents, wetting        agents, fertilizers, and combinations thereof.    -   5. The insecticidal composition of embodiment 1, formulated as a        dry solid.    -   6. The insecticidal composition of embodiment 1, formulated as a        liquid.    -   7. The insecticidal composition of embodiment 1, formulated for        foliar application.    -   8. The insecticidal composition of embodiment 1, formulated for        in-furrow application.    -   9. The insecticidal composition of embodiment 1, formulated as a        seed coating or seed treatment.    -   10. The insecticidal composition of embodiment 1, further        comprising: at least one additional pesticidal compound.    -   11. The insecticidal composition of embodiment 1, further        comprising: at least one additional pesticidal compound selected        from the group consisting of: a Monalysin protein, Pseudomonas        insecticidal protein, Cry protein, Cyt protein, vegetative        insecticidal protein, toxin complex protein, and any combination        thereof.    -   12. The insecticidal composition of embodiment 1, further        comprising: at least one additional herbicidal compound.    -   13. A method of killing a target pest, comprising: applying to        said target pest the insecticidal composition of embodiment 1.    -   14. A method of killing a target pest, comprising: applying to a        locus the insecticidal composition of embodiment 1, wherein said        target pest comes into contact with said locus.    -   15. A method of killing a target pest, comprising: applying to a        crop the insecticidal composition of embodiment 1, wherein said        target pest comes into contact with said crop.    -   16. A method of killing a target pest, comprising: applying to a        crop the insecticidal composition of embodiment 1, wherein said        target pest comes into contact with said crop, and said target        pest is a member of the Order Coleoptera, Diptera, Hymenoptera,        Lepidoptera, Hemiptera, Orthroptera, Thysanoptera, or        Dermaptera.

Cell Lysate

-   -   1. Cell lysate, comprising: an insecticidal protein comprising        an amino acid sequence with at least about 80% sequence identity        to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ        ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID        NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO:        26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34,        SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ        ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID        NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO:        60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68,        SEQ ID NO: 70, or SEQ ID NO: 72.    -   2. The cell lysate of embodiment 1, comprising: an insecticidal        protein comprising an amino acid sequence with at least about        90% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO:        6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14,        SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ        ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID        NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO:        40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48,        SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ        ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID        NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, or SEQ ID NO: 72.    -   3. The cell lysate of embodiment 1, comprising: an insecticidal        protein comprising an amino acid sequence with at least about        95% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO:        6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14,        SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ        ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID        NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO:        40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48,        SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ        ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID        NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, or SEQ ID NO: 72.    -   4. The cell lysate of embodiment 1, comprising: an insecticidal        protein comprising an amino acid sequence with at least about        99% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO:        6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14,        SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ        ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID        NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO:        40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48,        SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ        ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID        NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, or SEQ ID NO: 72.    -   5. The cell lysate of embodiment 1, comprising: an insecticidal        protein comprising an amino acid sequence selected from SEQ ID        NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,        SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ        ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID        NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO:        36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44,        SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ        ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID        NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO:        70, or SEQ ID NO: 72.    -   6. A method of killing a target pest, comprising: applying to        said target pest the cell lysate of embodiment 1.        Methods of Killing Pests with a Natural Microbe Expressing the        Insecticidal Protein    -   1. A method of killing a target pest, comprising: applying to        said target pest a host cell that expresses a polynucleotide        encoding a polypeptide having at least about 80%, 81%, 82%, 83%,        84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, 99%, or greater, sequence identity to an amino acid        sequence selected from the group consisting of: SEQ ID NO: 2,        SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID        NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:        20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28,        SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ        ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID        NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO:        54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62,        SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, and        SEQ ID NO: 72.    -   2. The method of embodiment 1, wherein the host cell expresses a        polynucleotide that encodes a polypeptide having an amino acid        sequence selected from the group consisting of: SEQ ID NO: 2,        SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID        NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:        20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28,        SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ        ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID        NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO:        54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62,        SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, and        SEQ ID NO: 72.    -   3. The method of embodiment 1, wherein the host cell is a        Prokaryotic host cell.    -   4. The method of embodiment 1, wherein the host cell naturally        expresses the polynucleotide.    -   5. The method of embodiment 1, wherein the host cell is from the        genus Pseudomonas.    -   6. The method of embodiment 1, wherein said target pest is a        member of the Order Coleoptera, Diptera, Hymenoptera,        Lepidoptera, Hemiptera, Orthroptera, Thysanoptera, or        Dermaptera.

Insecticidal Protein Discovery Platform

-   -   1. A method for constructing a genomic library, enriched for DNA        from Pseudomonas encoding insecticidal proteins, comprising:        -   a. providing an initial sample comprising one or more            microorganisms;        -   b. exposing the initial sample to a solid nutrient limiting            media that enriches for growth of species from the genus            Pseudomonas, which results in a subsequent sample enriched            for Pseudomonas sp.;        -   c. isolating DNA from the subsequent enriched sample;        -   d. extracting DNA from the isolated DNA and performing            degenerate PCR with primers selected to amplify target            insecticidal protein genes;        -   e. cloning the PCR-amplified DNA into a plasmid; and        -   f. sequencing the cloned DNA from said plasmid.    -   2. The method of embodiment 1, further comprising: assembling        the sequenced DNA into a genomic library.    -   3. The method of embodiment 1, further comprising: identifying        insecticidal protein genes within the sequenced DNA.    -   4. The method of embodiment 1, further comprising: identifying        insecticidal protein genes within the sequenced DNA, wherein        said identified insecticidal protein genes are unknown.    -   5. The method of embodiment 1, further comprising: utilizing a        Hidden Markov model to identify insecticidal protein genes        within the sequenced DNA.    -   6. The method of embodiment 1, further comprising: identifying        insecticidal protein genes within the sequenced DNA, wherein        said identified insecticidal protein genes are selected from the        group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17,        19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,        51, 53, 55, 57, 59, 61, 63, 65, 67, 69, and 71.    -   7. The method of embodiment 1, further comprising: identifying        insecticidal protein genes within the sequenced DNA, wherein        said identified insecticidal protein genes encode a protein        selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10,        12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,        44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, and 72.    -   8. The method of embodiment 1, wherein the primers are selected        to amplify target insecticidal protein genes that encode a        protein with at least 50% sequence identity to SEQ ID NO: 39.    -   9. The method of embodiment 1, wherein the initial sample is        from soil.    -   10. An insecticidal genomic library enriched for DNA from        Pseudomonas encoding insecticidal proteins, as constructed by        the method of embodiment 1.

HMM Model Proteins

-   -   1. An insecticidal protein, comprising: a) an amino acid        sequence that scores at or above a bit score of 521.5; and/or b)        an amino acid sequence that matches at an E-value of less than        or equal to 7.9e-161, when scored or matched using the HMI in        Table 6.

An insecticidal protein encoding nucleic acid, as set forth in Table 3,or an insecticidal protein having an amino acid sequence, as set forthin Table 3, are embodiments of the present disclosure, as well asmethods of using the same for the control of insect pests, and methodsof discovering same.

INCORPORATION BY REFERENCE

All references, articles, publications, patents, patent publications,and patent applications cited herein are incorporated by reference intheir entireties for all purposes. However, mention of any reference,article, publication, patent, patent publication, and patent applicationcited herein is not, and should not be taken as an acknowledgment or anyform of suggestion that they constitute valid prior art or form part ofthe common general knowledge in any country in the world.

What is claimed is:
 1. An insecticidal composition, comprising: a) aninsecticidal protein having an amino acid sequence (i) with at least 97%sequence identity to a protein with the amino acid sequence of SEQ IDNO: 20; or (ii) with at least 96% sequence identity to a protein with anamino acid sequence selected from the group consisting of: SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:18, and SEQ ID NO: 22; or (iii) with at least 94% sequence identity to aprotein with the amino acid sequence of SEQ ID NO: 8; and b) anagriculturally acceptable carrier.
 2. The insecticidal composition ofclaim 1, wherein said insecticidal protein has an amino acid sequencewith at least about 97% sequence identity to a protein with an aminoacid sequence selected from the group consisting of: SEQ ID NO: 2, SEQID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQID NO: 18, and SEQ ID NO:
 22. 3. The insecticidal composition of claim1, wherein said insecticidal protein has an amino acid sequence with atleast about 98% sequence identity to a protein with an amino acidsequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:18, SEQ ID NO: 20, and SEQ ID NO:
 22. 4. The insecticidal composition ofclaim 1, wherein said insecticidal protein has an amino acid sequencewith at least about 99% sequence identity to a protein with an aminoacid sequence selected from the group consisting of: SEQ ID NO: 2, SEQID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQID NO: 18, SEQ ID NO: 20, and SEQ ID NO:
 22. 5. The insecticidalcomposition of claim 1, wherein said insecticidal protein has an aminoacid sequence selected from the group consisting of: SEQ ID NO: 2, SEQID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQID NO: 18, SEQ ID NO: 20, and SEQ ID NO:
 22. 6. The insecticidalcomposition of claim 1, wherein said isolated insecticidal protein ispresent in an insecticidally effective amount.
 7. The insecticidalcomposition of claim 1, wherein said agriculturally acceptable carrieris selected from the group consisting of: adjuvants, inert components,dispersants, surfactants, sticking agents, tackifiers, binders, naturalor regenerated mineral substances, solvents, wetting agents,fertilizers, and combinations thereof.
 8. The insecticidal compositionof claim 1, formulated as a dry solid.
 9. The insecticidal compositionof claim 1, formulated as a liquid.
 10. The insecticidal composition ofclaim 1, formulated for foliar application.
 11. The insecticidalcomposition of claim 1, formulated for in-furrow application.
 12. Theinsecticidal composition of claim 1, formulated as a seed coating orseed treatment.
 13. The insecticidal composition of claim 1, furthercomprising an additional pesticidal compound.
 14. The insecticidalcomposition of claim 13, wherein the additional pesticidal compound isselected from the group consisting of: a Monalysin protein, aPseudomonas insecticidal protein, Cry protein, Cyt protein, vegetativeinsecticidal protein, toxin complex protein, and any combinationthereof.
 15. The insecticidal composition of claim 1, further comprisingan herbicidal compound.
 16. A method of killing a target pest,comprising: applying to said target pest the insecticidal composition ofclaim
 1. 17. A method of killing a target pest, comprising: applying toa locus the insecticidal composition of claim 1, wherein said targetpest comes into contact with said locus.
 18. The method of claim 17,wherein the locus is a crop.
 19. The method of claim 17, wherein saidtarget pest is a member of the order Coleoptera, Diptera, Hymenoptera,Lepidoptera, Hemiptera, Orthroptera, Thysanoptera, or Dermaptera.
 20. Acell lysate, comprising: an insecticidal protein having an amino acidsequence (i) with at least 97% sequence identity to a protein with theamino acid sequence of SEQ ID NO: 20; or (ii) with at least 96% sequenceidentity to a protein with an amino acid sequence selected from thegroup consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 12, SEQ IDNO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 22; or (iii) withat least 94% sequence identity to a protein with the amino acid sequenceof SEQ ID NO:
 8. 21. The cell lysate of claim 20, wherein saidinsecticidal protein has an amino acid sequence with at least about 97%sequence identity to a protein with an amino acid sequence selected fromthe group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8, SEQID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO:22.
 22. The cell lysate of claim 20, wherein said insecticidal proteinhas an amino acid sequence with at least about 98% sequence identity toa protein with an amino acid sequence selected from the group consistingof: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO:
 22. 23.The cell lysate of claim 20, wherein said insecticidal protein has anamino acid sequence with at least about 99% sequence identity to aprotein with an amino acid sequence selected from the group consistingof: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO:
 22. 24.The cell lysate of claim 20, wherein said insecticidal protein has anamino acid sequence selected from the group consisting of: SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16,SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO:
 22. 25. A method of killinga target pest, comprising: applying to said target pest the cell lysateof claim 20; or applying to a locus the cell lysate of claim 20, whereinsaid target pest comes into contact with said locus.
 26. The method ofclaim 25, wherein the locus is a crop.
 27. The method of claim 25,wherein said target pest is a member of the order Coleoptera, Diptera,Hymenoptera, Lepidoptera, Hemiptera, Orthroptera, Thysanoptera, orDermaptera.
 28. A method of killing a target pest, comprising: applyingto said target pest a host cell that expresses a polynucleotide encodinga polypeptide having an amino acid sequence (i) with at least 97%sequence identity to a protein with the amino acid sequence of SEQ IDNO: 20; or (ii) with at least 96% sequence identity to a protein with anamino acid sequence selected from the group consisting of: SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:18, and SEQ ID NO: 22; or (iii) with at least 94% sequence identity to aprotein with the amino acid sequence of SEQ ID NO:
 8. 29. The method ofclaim 28, wherein said polypeptide has an amino acid sequence with atleast about 97% sequence identity to a protein with an amino acidsequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:18, and SEQ ID NO:
 22. 30. The method of claim 28, wherein saidpolypeptide has an amino acid sequence with at least about 98% sequenceidentity to a protein with an amino acid sequence selected from thegroup consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8, SEQ IDNO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, andSEQ ID NO:
 22. 31. The method of claim 28, wherein said polypeptide hasan amino acid sequence with at least about 99% sequence identity to aprotein with an amino acid sequence selected from the group consistingof: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO:
 22. 32.The method of claim 28, wherein said polypeptide has an amino acidsequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:18, SEQ ID NO: 20, and SEQ ID NO:
 22. 33. The method of claim 28,wherein the host cell is a Prokaryotic host cell.
 34. The method ofclaim 28, wherein the host cell naturally expresses the polynucleotide.35. The method of claim 28, wherein the host cell is from the genusPseudomonas.
 36. The method of claim 28, wherein said target pest is amember of the order Coleoptera, Diptera, Hymenoptera, Lepidoptera,Hemiptera, Orthroptera, Thysanoptera, or Dermaptera.